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TURMERIC AND ITS ALLIES
MARLINA ARDIYANI
2003
"Behold, in the creation of the heavens and the earth, in the alteration
of night and day, in the sailing of the ships through the ocean for the
profit of mankind, in the rain which Allah sends down from the skies
and the life which He gives therewith to an earth that is dead, in the
beasts of all kinds that he scatters through the earth, in the change of
the winds and the clouds which they trail like their slaves between the
sky and the earth, here indeed are signs for a people that are wise".
(Al-Ba qarah: 164)
For my husband, my daughter, my son,
and my family
thanks for your support, love,
and sacrifice
ABSTRACT
The genus Curcurna L. comprises more than 80 species distributed from India, Burma,
Thailand, Southeast Asia, to Queensland and the Pacific Islands. One of its two
subgenera, subgenus Curcuma (Baker) K. Schum., contains many morphologically
highly similar polyploid taxa, which have been treated at specific level by previous
authors, contributing to taxonomic confusion in the genus. I have studied this problem
using a sample of the genus which includes all the Javanese Curcuina (16 species) as
well as 5 species from India, 7 species from Thailand and one from Australian/New
Guinea. The closely related genera Smithairis W.J. Kress & K. Larsen and Siahlianihus
0. Kuntze have also been sampled. Roscoea Sm. and Caulleya (Royle ex Benth.)
Hook.f. were used as outgroups.
A phylogenetic study of Curcuina was carried out using 35 gross morphological
characters and molecular data from the nuclear ribosomal DNA (ITS; internal
transcribed spacer region) and plastid DNA of the ItnL-F region. The tree that resulted
from the morphological approach was not well resolved, especially in subgenus
Curcuma. The molecular data gave a more resolved tree. Both trees are almost
congruent except that C. aurantiaca Zijp and C. qf ausiralasica Hook.f., which were
placed between C. econiala Craib and the C. ihorelii Gagnep. dade in the molecular
analyses, were shifted to nodes between C. roscoeana Wall. and C. peliolala Roxb. in
the tree constructed from morphological data. Molecular phylogenetic analysis shows
that Curcuina is not monophyletic. Smiiha/ri.s and Stahlianthus are nested within
Curcuma and need to be transferred to Curcuma to make it monophyletic. Subgenera
Hitcheniopsis ( Baker) K. Schum. and Curcurna are phylogenetically distinct. Subgenus
Curcunia is monophyletic with good support in the ITS data (BS=95; D1+4). Current
attempted sectional level classification within subgenus Curcuma should be abandoned,
as it is mainly based on inflorescence position, which is homoplasious. Analysis of ITS
polymorphisms in subgenus Curcuina reveals indel (one to four bp) polymorphisms
within an individual, suggesting possible hybrid origin for some species in subgenus
Curcuma.
Anatomical study of epidermal characters, leaf transverse section and SEM of seeds
revealed patterns of similarity among species. Principal Component Analysis of
epidermal and stomatal cell measurements did not reveal obvious clusters.
Floral diversity in Curcuma was examined using Principal Component Analysis.
Three floral types, ie. complex, small, and simple flowers, suggest three putative
pollination syndromes. Mapping morphological characters (especially those characters
used in the existing classification) onto the molecular tree gave some insight to the
evolutionary history of Curcurna. Mapping the floral types show that the simple flower
in subgenus Curcuma was probably derived from the more complex flower
characteristic of subgenus Hitcheniop.sis.
Isozyme electrophoresis reveals isozyme polymorphism in populations of C.
colorata Valeton, C. heyneana Valeton & Zijp, C. longa L., and C. zanihorrhiza Roxb.
The pattern of polymorphism is interpreted as possibly indicating a multiple origin of the
'species'. Chromosome count results from Curcuina subgenus Curcuina were mostly
triploid 2n63; while counts for subgenus Hitcheniopsis was different (eg. C. tho re/li;
2n=c.38). Revision of Javanese Curcuma is presented with a proposed new
classification, key, and descriptions.
ACKNOWLEDGEMENTS
First of all, I would like to thank to Allah, God of the 'Alameen (the whole
universe), for all of His blessings. It was Allah who has opened a broad way for me so
Quentin Cronk and Mark Newman, for their guidance and help, huge understanding,
would like to thank them all: Andy and Pat in the Research Glasshouse who have helped
me repotting Curcumas and looking after them; Fiona Inches, Andrea Fowler, and
Stephan Heifer from Quarantine section; Kwiton Jong who has guided me patiently
throughout my cytological work; Ruth Hollands who has taught me on isozyme work,
and helped me with other lab work; Michelle Hollingsworth who has guided me and
taught me every little useful things in molecular lab; Micha Bayer for kindly gave me
access to his computer imaging system; Debby White for her patience in photographing
Curcurnas; Frieda Christie for teaching me the SEM work; Pete Hollingsworth for short
suggestion on the anatomical work; Michael Möller for German translation and Hans
Sluirnan for Dutch translation; John Mood for sending me leaves sample ofCurcumas;
Martin Pullan for his help on Pandora database; the very kind library staff and other staff
in the Royal Botanic Edinburgh especially to Douglas McKean, Phil Lusby, Helen Hoy,
Marisa Main, Maureen Warwick, Mary Mendum, Rita Calder; as well as staff in the
I declare that this thesis has been composed by myself and that it contains no
material which has been accepted for the award of a degree in any university. All
quotations have been distinguished by quotation marks and other sources have been
clearly acknowledged.
Iv
CONTENTS IN BRIEF
CHAPTER 1: INTRODUCTION 1
UT
TABLE OF CONTENTS
ABSTRACT
ACKNOWLEDGEMENTS
DECLARATION iv
CONTENTS IN BRIEF v
TABLE OF CONTENTS vi
LIST OF FIGURES xiii
LIST OF TABLES xvii
LIST OF APPENDICES xviii
CHAPTER 1: INTRODUCTION I
Vi
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR
DATA FROM NUCLEAR TRANSCRIBED SPACER (RIBOSOMAL
DNA) AND TRNL-F PLASTID DNA SEQUENCES 29
2.1 INTRODUCTION 29
2.1.1 Internal Transcribed Spacer (ITS) region of ribosomal DNA 29
2.1.2 Functions of the ITS 30
2.1.3 Advantages and disadvantages of using the ITS 31
2.4 MATERIALS 36
2.4.1 Ingroup and outgroup taxa 36
2.6 RESULTS 55
2.6.1 Total genomic DNA extraction 55
2.6.2 Gene amplification and purification of PCR products 56
2.6.3 Cycle sequencing and automated DNA sequencing 59
2.6.4 Sequence analysis 61
2.6.5 Phylogenetic analysis 62
2.7 DISCUSSION 72
2.7.1 PHYLOGENETIC RELATIONSHIPS WITHIN CURCUMA AND
BETWEEN CURCUMA AND ITS OUTGROUP 72
2.7.1.1 Basal dade - Stahlianthus involucratus, Smithatris
supraneanae, and C. ecomata 72
2.7.1.2 The C. aurantiacaclade 73
2.7.1.3 The C. paiviflora dade 73
2.7.1.4 The subgenus Curcuma dade 74
2.7.1.5 C. roscoeana and C. petiolata 76
VII
CHAPTER 3: PHYLOGENETIC STUDY USING
MORPHOLOGICAL DATA 78
3.1 INTRODUCTION 78
3.1.1 Characters used in the existing infrageneric classification of
Curcuma 78
3.1.1.1 Rhizomes 78
3.1.1.2 Leaves 79
3.1.1.3 Ligules 79
3.1.1.4 Fertile bracts 79
3.1.1.5 Coma bracts 80
3.1.1.6 Staminode 80
3.1.1.7 Anther 80
3.1 .2 Are there any other characters defining infrageneric classification in
Curcuma?: a search for them 81
3.1.2.1 Leaf anatomy 81
3.1.2.2 Stigma 81
3.1.2.3 Flower 81
3.1.2.4 Seeds 82
3.2 MATERIALS 82
3.2.1 Origin of plant materials, outgroup and ingroup taxa 76
Viii
3.4 RESULTS 55
3.4.1 CODED MORPHOLOGICAL CHARACTERS AND PHYLOGENETIC
ANALYSIS 85
3.4.1.1 Gross morphological characters (external morphology and light
microscopy) 85
3.4.1.1.1 The habit 85
3.4.1.1.2 Rhizome structure 88
3.4.1.1.3 Colour of internal rhizome 90
3.4.1.1.4 Smell of rhizome 93
3.4.1.1.5 Leaf shape 93
3.4.1.1.6 Shape of ligule 93
3.4.1.1.7 Colour of midrib 95
3.4.1.1.8 Colour of sheath 95
3.4.1.1.9 Purple flush on leaves 95
3.4.1.1.10 Position of inflorescence 99
3.4.1.1.11 Shape of bracts 101
3.4.1.1.12 Length of coma compared to fertile bracts 101
3.4.1.1.13 Colour of coma and fertile bracts 101
3.4.1.1.14 Flower structure 105
3.4.1.1.15 Hair on ovary 105
3.4.1.1.16 Shape of petals 105
3.4.1.1.17 Colour of petals 105
3.4.1.1.18 Hair on dorsal petal 107
3.4.1.1.19 Cucullate on dorsal petal 107
3.4.1.1.20 Shape of labellum 107
3.4.1.1.21 Colour oflabellum 107
3.4.1.1.22 Hair on labellum blade 109
3.4.1.1.23 Hair on the middle band of the labellum 109
3.4.1.1.24 Shape of lateral staminodes 109
3.4.1.1.25 Arrangement of lateral staminodes 109
3.4.1.1.26 Colour of lateral staminodes 111
3.4.1.1.27 Groove on lateral staminodes 111
3.4.1.1.28 Patch of granules at apex of lateral staminodes 111
3.4.1.1.29 Length of anther 111
3.4.1.1.30 Spur on anther 114
3.4.1.1.31 Crest on anther 114
3.4.1.1.32 Anther dehiscence 114
3.4.1.1.33 Stigma type 114
3.4.1.1.34 Stylar growth 114
3.4.1.1.35 Ring of hair on corolla tube 115
ix
3.4.3 MORPHOMETRIC OF EPIDERMIS IKIS]
x
CHAPTER 4: EVOLUTION OF FLORAL DIVERSITY
IN CURCUMA 147
Xi
6.2.5 Stain preparation 184
6.2.6 Gel slicing and staining 185
REFERENCES 266
XII
LIST OF FIGURES
CHAPTER 1
Figure 1.1 Cladogram showing relationships of Zingiberales to monocots 2
Figure 1.2 Map of distribution of Zingiberaceae and Curcuma 6
Figure 1.3 Flower structure of the tribes in Zingiberaceae 11
Figure 1.4 Illustration of Curcuma subgenus Curcuma and subgenus
Hitcheniopsis. 13
Figure 1.5 Diagram of compound cincinnus flower 14
CHAPTER 2
Figure 2.1 Repeat units of the nuclear ribosomal DNA and the organizationof
the ITS region. 34
Figure 2.2 Chloroplast DNA 34
Figure 2.3 The chloroplast DNA region between the trnT (UGU) and the trnF
(GAA) genes. 35
Figure 2.4 Flowchart of the methods used in the molecular study. 40
Figure 2.5 Flowchart to show simplified method to extract the nucleic acids. 42
Figure 2.6 General protocol of the polymerase chain reaction for amplifying
DNA (after Oste 1988 in Avise 1994) 46
Figure 2.7 The QlAquick spin purification procedure (modified from QlAquick
Spin Handbook) 48
Figure 2.8 AutoAssembler assemble the complementary strands automatically
from the whole four forward and reverse sequences. 51
Figure 2.9 Gel electrophoresis from total genomic DNA extraction. 55
Figure 2.10 Gel electrophoresis result from PCR of ITS and region between
trnL (UAA) 5' exon and trnF (GAA). 57
Figure 2.11 Gel electrophoresis result from PCR of region between trnL (UAA)
5' exon and trnF (GAA). 58
Figure 2.12 Gel electrophoresis result from PCR of ITS region. 59
Figure 2.13 The electropherogram an automated DNA sequencing of ITS2
region of C. aurantiaca showing clean non-polymorphic sequence. 60
Figure 2.14 Strict consensus tree obtained from 613 equally most parsimonious
trees of length 280 steps resulted from equally weighted parsimony analysis of
the combined ITS1 and ITS2 data with gaps treated as missing data (C10.7143;
RI=0.8238; RC0.5884). 66
Figure 2.15 Strict consensus tree obtained from 48 equally most parsimonious
trees of length 322 steps resulted from equally weighted parsimony analysis of
the combined ITSI and ITS2 data with indels treated as coded present absent
(Cl=0.6801; RI=0.8147; RC=0.5541). 67
XIII
Figure 2.16 One most parsimonius tree of length 29 steps resulted from equally
weighted parsimony analysis of the chloroplast DNA between trnL (UAA) 3' exon
and trnF (GAA) data with gaps treated as missing data (Cl1 .000; RI=1 .000;
RC=1.000). 68
Figure 2.17 Strict consensus tree obtained from 55 equally most parsimonious
trees of length 53 steps resulted from equally weighted parsimony analysis of
the chioroplast DNA between trnL (UAA) 3' exon and trnF (GAA) data with
indels treated as coded present absent (Cl0.7531; Rl0.7692; RC=0.5793). 69
Figure 2.18 Strict consensus tree obtained from 9 equally most parsimonious
trees of length 243 steps resulted from equally weighted parsimony analysis of
the combined ITS and chloroplast DNA between trnL (UAA) 3' exon and trnF
(GAA) data with gaps treated as missing data (Cl=0.6775; Rl=0.7265;
RC0.4922). 70
Figure 2.19 Strict consensus tree obtained from 8 equally most parsimonious
trees of length 307 steps resulted from equally weighted parsimony analysis of
the combined ITS and chloroplast DNA between trnL (UAA) 3' exon and trnF
(GAA) data with indels treated as coded present absent (Cl=0.6775; Rl=0.7265;
RC=0.4922). 71
CHAPTER 3
Figure 3.1 The habit of Curcuma. 87
Figure 3.2 Rhizome structure in Curcuma. 89
Figure 3.3 Colour of rhizome section in Curcuma subgenus Curcuma. 91
Figure 3.4 Variation of leaf shape in individual species of C. aeruginosa 94
Figure 3.5 Ligule shape in Curcuma 96
Figure 3.6 Colour of midrib in Curcuma 96
Figure 3.7 Colour of sheath in Curcuma. 97
Figure 3.8 Variation of stretches on leaves in Curcuma. 97
Figure 3.9 Position of inflorescence in Curcuma. 98
Figure 3.10 Variation of bract shape and colour in Curcuma. 102
Figure 3.11 Variation of flower shape in Curcuma 104
Figure 3.12 Variation of petal shape in Curcuma 106
Figure 3.13 Petal colours in Curcuma 106
Figure 3.14 Labellum shape, and hair on the middle band of the labellum in
Curcuma 108
Figure 3.15Shape and orientation of lateral staminodes in Curcuma. 110
Figure 3.16Anther of Curcuma. 112
Figure 3.17Anther of Curcuma from the back. 113
Figure 3.18Stigma type in Curcuma. 115
Figure 3.19Strict consensus tree obtained from 1000 equally most
parsimonious trees of length 75 steps resulted from equally weighted parsimony
analysis of morphological (C10.5789; Ri=0.8621; RC0.4991). 121
Figure 3.20 Adaxial epidermis in some representative Curcuma spp. 124
Figure 3.21 Abaxial epidermis in some representative Curcuma spp. 125
Figure 3.22 Abaxial epidermis of C. mangga and C. amada. 126
xlv
Figure 3.23 Leaf transverse section. 127
Figure 3.24 SEM of seed surface. 129
Figure 3.25 Principal component plot of Curcuma. 132
Figure 3.26 Mapping the habit (A), rhizome structure (B), colour of internal
rhizome (C), and shape of ligule (D) onto molecular tree. 134
Figure 3.27 Mapping position of inflorescence (A), hair on the midddle band of
the labellum (B), arrangment of lateral staminodes (C) onto molecular tree, and
groove on lateral staminodes (D). 141
Figure 3.28 Mapping spur on anther (A) and crest on anther (B) onto molecular
tree. 142
CHAPTER 4
Figure 4.1 Measurement of flowers for morphometric analysis. 153
Figure 4.2 Eigenvectors of the first and the second axes for the 28 floral
characteristics 155
Figure 4.3 Floral types of Curcuma 157
Figure 4.4 Principal component plot of Curcuma. 158
Figure 4.5 Principal component plot of Curcuma. 159
Figure 4.6 Principal component plot of Curcuma. 160
Figure 4.7 The mapping of flower type onto molecular tree. 162
CHAPTER 5
Figure 5.1 Flowchart of chromosome study. 165
Figure 5.2 Chromosomes of C. aeruginosa. 172
Figure 5.3 Chromosomes of C. heyneana. 172
Figure 5.4 Chromosomes of C. mangga 173
Figure 5.5 Chromosomes of C. thorelii. 173
Figure 5.6 Chromosomes of C. zanthorrhiza. 174
Figure 5.7 Chromosomes of C. zedoaria. 174
CHAPTER 6
Figure 6.1 Diagrammatic representation of isozyme loci and allozymes. 176
Figure 6.2 General protocol for protein-electrophoretic surveys (modified from
Avise 1994) 180
Figure 6.3 Cutting and loading the gel (Murphy etal. 1996). 182
Figure 6.4 Horizontal starch gel apparatus during electrophoresis (Murphy et al.
1996). 183
Figure 6.5 Slicing the gel for staining (Murphy etal. 1996). 186
Figure 6.6 Isozyme phenotypes for PGI, PGM, and MDH. 189
CHAPTER 7
Figure 7.1 Electropherogram 193
Figure 7.2 Strict consensus tree obtained from 1000 equally most parsimonious
trees of length 167 steps resulted from equally weighted parsimony analysis of
1T52 data of non- and polymorphic Curcuma and the outgroups, with all sites
analysed (Cl=0.677; Rl=0.830; RC0.562). 199
xv
Figure 7.3 Strict consensus tree obtained from 112 equally most parsimonious
trees of length 147 steps resulted from equally weighted parsimony analysis of
ITS2 data of non- and polymorphic Curcuma and the outgroups, with indel
polymorphic sites excluded (Cl=0.701; Rl=0.835; RC=0.585). 200
Figure 7.4 Strict consensus tree obtained from 1000 equally most parsimonious
trees of length 163 steps resulted from equally weighted parsimony analysis of
ITS2 data of non- and polymorphic Curcuma and the outgroups, with indel
polymorphic sites coded as present or /and absent (C10.693; Rl=0.828;
RC=0.574). 201
CHAPTER 8
Figure 8.1 Index of picture to use in Figure 8.2 216
Figure 8.2 Illustration of Javanese Curcuma. 218
CHAPTER 9
Figure 9.1 Trees constructed from molecular data (the left), morphological
data(the middle), and combined both molecular and morphological data (the
right) 220
xvi
LIST OF TABLES
CHAPTER 1
Table 1.1 Systems of classification of Zingiberales 3
Table 1.2 Characteristics and tribes of Zingiberaceae 9
Table 1.3 Classification of Curcuma. 18
Table 1.4 Original description of sections or subgenera in Curcuma. 20
CHAPTER 2
Table 2.1 50 accessions representing 31 taxa of used in
the molecular study (ITS and trnL-F). 38
Table 2.2 Primers used in PCR and cycle sequencing. 49
Table 2.3 Cladistic terminology and their definition or function,
used in phylogenetic analysis. 54
Table 2.4 Sequence characteristics of ITS1 and ITS2 regions of 28 taxa of
Zingiberaceae. 64
Table 2.5 Sequence characteristics of chloroplast regions between trnL (UAA)
5' exon and trnF (GAA) of 25 taxa of Zingiberaceae. 65
CHAPTER 3
Table 3.1 Colour of the internal rhizome of Curcuma. 92
Table 3.2 Frequent inflorescence position in Curcuma spp. 100
Table 3.3 Character coding for morphological data of Curcuma spp. 116
Table 3.4 Variance extracted, first 8 axes (of morphometric of epidermal
characters) 131
CHAPTER 4
Table 4.1 Curcuma taxa used in this study 150
Table 4.2 Characters scored for morphometric analysis. 152
Table 4.3 Variance extracted, first 10 axes. 164
CHAPTER 5
Table 5.1 List of materials used in the study. 156
Table 5.2 Results of chromosome counts in this study. 169
Table 5.3 Chromosome counts from this study and from literature. 170
CHAPTER 6
Table 6.1 Curcuma taxa used in this study. 179
CHAPTER 7
Table 7.1 Indel polymorphisms of ITS1 region 195
Table 7.2 Indel polymorphisms of ITS2 region. 196
Table 7.3 Descriptive statistics reflecting the amount of phylogenetic signal
under different condition of parsimony analysis for data on Appendix 6. 202a
xvii
CHAPTER 8
Table 8.1 Taxonomic treatment of Javanese Curcuma. 203
Table 8.2 The original description of Linnaeus Species Plantarum 1753 204
Table 8.3 Summary of the history of typification of Curcuma 205
LIST OF APPENDICES
APPENDIX 2 Sequence data matrix (displayed from 5' to 3') of aligned ITS1
and ITS2 regions of 28 taxa of Zingiberaceae. 226
APPENDIX 3 Sequence data matrix (displayed from 5' to 3') of aligned ITSI
and ITS2 regions of 28 taxa of Zingiberaceae. 232
APPENDIX 6 Sequence data matrix (displayed from 5' to 3') of aligned ITS2
region of 32 accessions representing 27 taxa of Zingiberaceae. 251
APPENDIX 7 Sequence data matrix (displayed from 5' to 3') of aligned 11S2
region of 32 accessions representing 27 taxa of Zingiberaceae after polymorphic
sequences combined. 257
xviii
CHAPTER 1: INTRODUCTION
correlated characters (Tomlinson 1962; Cronquist 1981; Dahlgren el al. 1985; Kress
1990), combined with the order Bromeliales, forms the subclass Zingiberidae Cronquist
in the class Liliopsida Cronquist or Monocotyledons (Cronquist 1978, 1981). In the
past, the order used to be called Scitamineae or Arillatae (Engler 1892). Recent study
(Kress etal. 200 1) using combined morphological and molecular data shows the
monophyly of the order Zingiberales which is placed on terminal in the cladogam among
highlighted five different systems and Kress proposed another system of classification.
The first system was that of Bentham & Hooker (1883) which recognized Scitamineae
Bentham & Hooker, Zingibereae Bentham & Hooker, Maranteae Bentham & Hooker,
and Canneae Bentham & Hooker. Then, in 1889, Petersen in Engler & Prantl, who gave
the rank Reihe to the Scitamineae, recognized the four tribes of Bentham & Hooker at
family rank. He divided the Musaceae A.L.Jussieu into two tribes, namely, Museae
Petersen and Heliconieae Petersen. The refinement and division was continued by
raising the Museae from tribal to subfamily rank, Musoideae K. Schum., and by raising
Streliizia W. Aiton from the tribe Museae to the subfamily Strelitzioideae K. Schum.
New ranks were given to the tribe Heliconieae K. Schum, and the subfamilies
Zingiberoideae K. Schum and Costoideae K. Schum. Subfamily Lowioideae K. Schum
was later included in the system (Schumann in Engler 1900, 1902, 1904; Winkler in
Engler & Prantl 1930; Loesener in Engler & Prantl 1930). Hutchinson (1934, 1959)
raised the Strelitzioideae to family level and separated it from the Musaceae resulting in
CHAPTER 1: INTRODUCTION
Marantaceae
Canaceae
Zingiberaceae
Costaceae
Heliconiaceae
Strelitziaceae
Low iaceae
Musaceae
Commelinaceae
Hanguanaceae
Commelinaceae
Pontederiaceae
Bromeliaceae
Poaceae
Amaryllidaceae
Liliaceae
Dioscoreaceae
Asparagaceae
Araceae
Aristolochiaceae
2
CHAPTER 1: INTRODUCTION
Petersen Schumann
Bentham & Hooker 1883 (Engler & Prantl 1889) (Engler 1900, 1902, 1904, 1912),
Winkler (Engler & Prantl 1930),
Loesener (Engler & Prantl 1930)
Family Scitamineae Reihe Scitamineae Order Scitamineae K. Schum.
Bentham & Hooker Petersen
Tribe 3. Canneae Bentham Family 3. Cannaceae A.L. Family 3. Cannaceae A.L. Jussieu
& Hooker Jussieu
(later Zingiberales)
Family 1. Musaceae A.L. Family I. Musaceae A.L.Jussieu Suborder 1. Musineae Kress
Family 5. Cannaceae A.L. Family 7. Cannaceae A.L. Jussieu Superfamily 2. Cannariae Kress
Jussieu
Family 7. Cannaceae A.L.
Jussieu
Family 6. Marantaceae Family 8. Marantaceae Petersen Family 8. Marantaceae Petersen
Petersen
CHAPTER 1: INTRODUCTION
six families in the order. Nakai (1941) raised the rank of the Costeae Meisner and
separated it from the Zingiberaceae Lindley. This was followed by Tomlinson (1962,
1969), who accepted Nakai's system after investigating the distribution of anatomical
characters in the order. Takhtajan (1980) also followed Nakai. Nakai also separated the
Heliconieae from the Musaceae and raised it to family level resulting in the
establishment of eight families in the order, which is commonly followed by modern
taxonomists and phylogeneticists (Kress 1990).
5
CHAPTER 1: INTRODUCTION
reaching temperate regions of the mountains of China, Burma, North India and the
Himalaya (Cowley 1982, Ngamriabsakul et al. 2000).
7
CHAPTER 1: INTRODUCTION
of, e.g. Zingiber, Alpinia, and Amomum, on the basis of the character of the lateral
staminodes which are very small or absent. However, if we consider Zingiber as having
petaloid lateral staminodes deeply adnate to the labellum, we will exclude it from the
tribe.
1-lolttum (1950) revised this by excluding the genus from Petersen's tribe
Zingibereae, shifting it to the tribe Hedychieae on the basis of the lateral staminodes that
are free from the labellum or deeply adnate to the labellum in Zingiber. His tribe
Hedychieae was not legitimate according to the International Code for Botanical
Nomenclature as it contains Zingiber which is the type name for the family, the order,
etc. (Burtt & Olatunji 1972).
After intensive study, Olatunji (1970), and Burtt & Olatunji (1972) proposed a
new tribe Zingibereae which includes Zingiber alone (see Table 1.2), leaving
anther-thecae and stigma protruding at top of these, anther crest if present flat in
Hedychieae; petiole swollen and pulvinus-like in Zingibereae vs. not swollen nor
pulvinus-like in Hedychieae; vascular bundle with collenchymatous sheath in
axis of the rhizome was first described by Weisse in 1931, 1933 (cited in Burtt &
Olantunji 1972).
However, the delimitation is still not wholly clear (Newman 1988) as some
species in one tribe possess characters that fit the criteria of another tribe. An example is
given here from Newman (1988). Gagnepainia K. Schum., which is the member of
CHAPTER 1: INTRODUCTION
Small or absent, Petaloid, free from Petaloid, free from Petaloid, adnate to
Lateral staminodes
never petaloid labellum labellum and labellum
sometimes connate
to filament
Globbeae, has a short and not long exserted filament. On the other hand, Hedychiuin,
which is a member of Hedychieae, has a long exserted filament. The question was also
faced when Smith (cited in Newman 1988) discovered a new genus of which most of the
characters fit the description of tribe Hedychieae but lacking lateral staminodes. This
It has been reported that there are more than 40 species in Thailand (Sirirugsa,
unpublished data), 31 species in India (Velayudhan etal. 1996) of which 12 are endemic
(Jain & Prakash 1995), and more than 20 species in Malesia.
word kurkum in Arabic means yellow, however turmeric in Arabic is Uruku's sufr or
Uruku's sabaghin or Carcumaa Avicenna. Kurkum is Persian according to Richardson,
Arabic in the dictionary of Golius and Meninski, Hebrew in Parkhurst lexicon, but
derived from the same source as the Sanscrit Cuncuma (not Curcuma), with the Greek
Crocos and Crocon and with the Latin Crocus and Crocum all denoting saffron.
Rumphius had already remarked on the affinity of these names (Roxburgh 1812). He
derives the name Curcum from a Chaldaic word, to wash or anoint (Graham 1839).
Can the word tell the place of origin of Curcu,na? It is hard to judge if the word
kurkum, which is more from West or South Asia, would relate to the place of origin of
CHAPTER 1: INTRODUCTION
11
CHAPTER 1: INTRODUCTION
Curcuma. In other places, for example Thailand to Malaysia and Indonesia, there is a
uniformity of people in calling turmeric as "yellow" in their own words. This is almost
similar to the case of people saying kurkum. However, there is a sign that kurkum
which may refer to turmeric has been long developed and cultivated in the region of
West and South Asia rather than in South East Asia. The history of turmeric will be
covered in more detail in Chapter Eight.
12
CHAPTER 1: INTRODUCTION
or
/ /
C
:4
13
CHAPTER 1: INTRODUCTION
bracteole
14
CHAPTER 1: INTRODUCTION
flower which is bisexual consists of the ovary which is inferior, hairy or not, unilocular
with basal placentation or trilocular with axile placentation. From the top of the ovary,
two slim cylindrical epigynous glands stand out. A few species lack these glands. The
stamen. The sepals are tubular and three-toothed (one rather deeply toothed and two
shallowly toothed). The petals are tubular or infundibular (long stalked-cupped shaped)
at the base and three-lobed at the apex. The dorsal petals are normally slightly bigger
than the lateral ones. The lateral staminodes consist of two petaloid structures adnate to
and flanking the stamen, fused at the base with the petals. These clasping staminodes
nearly hide the stamen and pistil. In some species, the lateral staminodes are free and
not clasping. The labellum which is petaloid obovate or almost circular or elongated,
slightly bibbed, and conspicuous is adnate at the base on the side with lateral
staminodes. It is formed by the inner whorl of the androecium. It has a thickened
longitudinal bar in the centre and in some species, the sides of the bar towards the base
are slightly erect. The side lobes are erect so as to form a wide channel whereas the
apical lobe is recurved or protruded. In some species, the side lobes are very short and
The filament is short and broad, constricted at the top and connected to the base
of the anther or the back of the connective making it versatile. The anther has two
thecae which are parallel with the connective at the back. They embrace the style that is
filiform and support it. The dehiscence is towards the front, while the back- and
sidewalls are very thick and fleshy. These fleshy walls end in short or long awlshaped
15
CHAPTER 1: INTRODUCTION
If1
CHAPTER 1: INTRODUCTION
Valeton in 1918, and recently by Velayudhan etal. in 1996. The classifications are
Roxburgh (1812) divided the genus into two sections, one with lateral spikes
which appear before or with the leaves, and the other with central spikes. He recorded
C. rubescens having a lateral inflorescence, but he also said "and sometimes from the
centre of the leaves ". It is therefore concluded that C. rubescens has both inflorescence
positions, lateral in May and then central in September (Roxburgh 1812).
Horaninow (1862) had the same idea as Roxburgh in dividing the genus into
sections Exantha (for species that produce a lateral inflorescence) and Mesantha (for
those species that produce a central inflorescence). However, he also added a new
section Amphiantha for species that produce both positions of inflorescence, such as C.
rubescens and C. decipiens.
Baker (1894) excluded section Amphianiha but accepted the two sections
Exaniha and Mesantha. He added a new section Hitcheniopsis (see Table 1.4). He
Eucurcuma and Hit cheniopsis. In Hitcheniopsis, he added that the bracts are adnate to
each other almost for their whole length and that the anther is spurless. His original
description is tabulated in Table 1.4. He put together sections Exantha and Mesaniha
17
CHAPTER 1: INTRODUCTION
Section
Stolonifera
Section
Amphianta
KV
CHAPTER 1: INTRODUCTION
Valeton stated that the proportion of the adnate part of the bracts to the free part is very
vague and useless in practice. He excluded subgenus Hitcheniopsis and coined a new
subgenus Paracurcuma which contained C. auranhiaca, C. petiolata, C. cordfolia, C.
,neraukensis, and C. latjfolia. His descriptions of subgenera Paracurcuma and
Eucurcuma are tabulated in Table 1.4.
Though Valeton criticized Schumann's use of length of adnation of the bracts,
one of his descriptions still mentions this feature. He says that, in subgenus
Paracurcuma, the bracts are adnate at least partly beyond the middle. This character is
not constant throughout the subgenus. C. alismati,folia has bracts that connect to each
other almost at the base, therefore this should match subgenus Eucurcuma according to
Valeton's concept. This is probably because he excluded C. alismabfolia from the
genus Curcuma. To bear in mind, his description is only based on several Curcuma
especially in Malesian region (C. aurantiaca, C. pehiolata, C. cordfolia, C. ineraukensis,
and C. lat?folia).
Velayudhan el al. (1996) proposed a new classification of Curcuma. Their
classification at subgeneric level is basically the same as the previous classification
which divided the genus into two subgenera, Curcuma and Paracurcuma. However, the
sectional level classification of subgenus Curcuma is very different. Previous authors
divided subgenus Curcuma into sections Exantha and Mesaniha mainly based on the
position of inflorescence. Velayudhan et al. proposed a sectional classification of
subgenus Curcuma on the basis of rhizome structures and the place on the tuber from
which the flower spikes arise (from tip or side). They proposed three sections and five
subsections. The first section is section Tuberosa for species in which the main root
stock or secondary root stock gives rise to sessile fingers. The second is section
Nonbuberosa for species lacking the sessile tubers but producing stipitate tubers in large
19
CHAPTER 1: INTRODUCTION
20
CHAPTER 1: INTRODUCTION
numbers. And the last is section Stolonfera for species which have stoloniferous tubers
arising from the rhizomes.
Section Tuberosa is subdivided into two subsections. The first is a subsection in
which flower spikes arise from the tips of sessile tubers of the preceding year's growth
during the off season (e.g. C. aeruginosa, C. zedoaria). The second is a subsection in
which flower spikes arise from the tip of the primary mother stock (primary mother
rhizome) or secondary stock (secondary mother rhizome) during the main growing
season, e.g. C. longa (Velayudhan et al. 1996). Sessile tubers, however, can grow to
form a mother rhizome which will produce another clone.
Section Nontuberosa, is divided into three subsections on the basis of the
position of the flower spike on the root stocks. In the first subsection the spikes arise
from the side of the root stocks; while, in the second subsection the spikes arise from the
tip of the root stocks. Third is a subsection in which the flower spikes arise both from
the tip and from the sides of the mother rhizomes in different seasons.
Section Tuberosa subsection I and section Nontuberosa subsection I (e.g. C.
zedoaria and C. neilgherrensis respectively) correspond with section Exaniha, while
section Tuberosa subsection 2 and section Nontuberosa subsection 2 (e.g. C. longa and
C. pseudomontana respectively) correspond with section Mesaniha (Velayudhan et al.
1996).
Valeton excluded some of Schumann's species that are included in his subgenus
Hiicheniopsis. They are C. roscoeana, C. parvflora, C. alismaifolia, C. sparganfolia,
C. gracillima, C. sylvestris, C. lanceolata, and C. kunstlerii. Together, according to him,
they "do not constitute a natural group " . He added that they have in common with
Curcurna their strobiliform inflorescence, but the structures of coma bracts, petals,
lateral staminodes, labellum, and anther, are very different from those of Curcuma.
The reason why he tends to exclude those species from Curcuma is as follows.
C. roscoeana was thought to be not Curcuma according to Valeton because it has:
21
CHAPTER 1: INTRODUCTION
"no coma, all bracts rigid, red, erect with a much recurved top (free, according to
Wallich, except at the broad base, adnate with the edges, according to Baker) ;dorsal
lobe not cucullate; staminodes not lobed, not connate with the filament (?); labellum
simple, not lobe, not concave, with two elevated lines in the centre, including a median
groove: anther terminal, articulate to the filament with a broad base, thecae distant
much shorter than the large connective which ends in a membranaceous, ciliate crest
C. parvifiora has "petals converge behind the stamen and slam inodes,
staminodesfreefromfilament and seem to be placed in exterior cycle; labellum patent,
recurved, not lobed not concave, without erect side parts and central bar, without a
median groove; anther terminal subarticulate and nutant with a broad base, very shori
thecae (opening by pores?), a very large fleshy connective prolonged into a
considerable crest. The violet lip radiating while lines shows more relation to
Gastrochilus ".
In C. alismalifolia the .. .... narrow parallel theca of the rather long crested
anther are attenuate at their base into a kind of spurs, and the connection with filament
is at the backside near the base, probably it is nutant"..
In C. sparganfolia the "bracts of the spike are quite free one from another;
anther with shortly pointed thecae is evidently terminal, siaminodes are free from
filament labellum entire
In C'.gracillima the "the bracts are all alike, erect with extant subacute tips;
anther terminal or versatile? ".
He added that no stylodes were seen in the four species above (C. parviflora, C.
alismatifolia, C. sparganij'olia, C.gracillima). My observation of those species, except
C. sparganfolia, agrees with Valeton that there are no epigynous glands on the top of
the ovary.
C. spivesiris has a "slender creeping rhizome; anther terminal with a recurved
violet crest, and emarginate lip with a yellow central spot and violet streaks on the
lobes ".
22
CHAPTER 1: INTRODUCTION
23
CHAPTER 1: INTRODUCTION
Baker (1894) also thought that the genus was difficult of determination. He put forward
a hypothesis that most species of section Exantha were probably just varieties.
Curcuma is a taxonomically difficult genus, a nightmare to plant hunters,
herbarium technician, and taxonomist (Mangaly & Sabu 1993). This is because they are
not easy to identify either from fresh materials or herbarium specimens. It is probable
that natural crossing to produce natural hybrids has occurred -probably in several of
Valeton's species (Holttum 1951). It is no wonder that Backer & Bakhuizen van den
Brink (1968) arrived at the conclusion that there were only two collective species within
subgenus Curcuma. However, their two collective species are based on the position of
inflorescence which is, again, not a good character as discussed previously. Their first
collective species is C. zedoaria sensu lab, which consists of infraspecific taxa C.
zedoaria sensu stricto (s.s.), C. heyneana, C. phaeocaulis, C. xanlhorrhiza, C.
aeruginosa, C. mangga, and C. sylvatica. The second collective species is C. viridiflora
s.l. (older name is probably C. montana) consisting of C. viridiflora s.s., C. longa, C.
purpurascens, C. coloraba, C. euchroma, C. brog, C. soloensis, and C. ochrorhiza. The
segregation at infraspecific level into "species" was on the basis of several characters but
all were colours, such as colour of rhizome, colour of leaves along the midrib, colour of
leaf sheath, colour of coma bracts, and colour of flower (Backer & Bakhuizen van den
Brink 1968). Therefore, the key is not very easy to use, for example the key to species
of C. brog and C. soloensis is as follows: rhizome citron-yellow in C. brog, versus
rhizome orange-yellow in C. soloensis (Backer & Bakhuizen van den Brink 1968).
Valeton is right in emphasising the importance of assigning colour using a colour chart.
He made use of the "Code des couleurs" by Klincsiek et Valette (1908) and
24
CHAPTER 1: INTRODUCTION
"Chromotaxia" of Saccardo.
Why are some species so similar to each other, disregarding colour? If we look
at some other species such as most of the Thai species of subgenus Hitcheniopsis, they
are fertile and we can easily distinguish them even if we fail to see the colour. We can
distinguish them by the shapes of their various organs such as bract and flower shape.
we shall very likely
However, if we look at all the sterile species of subgenus Curcuma,
arrive at the same conclusion as Backer & Bakhuizen van den Brink. Herbarium
specimens are therefore useless unless the colour is recorded precisely or the smell of
may be divided into tribes, a large genus into sections, large sections in series, etc. If
that is not enough, one can always create additional ranks immediately below any or all
of the principal or secondary ranks by adding the prefix "sub-" to the rank concerned.
For example, subfamily is a rank immediately below a family but above a tribe.
Subgenera is a rank immediately below a genus. Similarly, one can insert ranks above
any of the recognized ranks, e.g., a superorder or superdivision. The advantages of these
secondary ranks are, for instance, to ease of reference and identification, to call attention
25
CHAPTER 1: INTRODUCTION
are difficult to study, and as the living materials are not all to hand, it will be very
difficult to achieve the goal. Fieldwork was carried out in Java to get living material and
add them to a living collection. Good living collections in the glasshouses of the Royal
Botanic Garden Edinburgh, allowed me to observe flowering of several species between
general results as to the subgeneric and sectional boundaries in the genus. As some
triploid sterile species from subgenus Curcuma are difficult to work out using
morphological investigation, a molecular study was also carried out as part of the
project. It is hoped that, by phylogenetic analysis using a different source of data, I
would be able to draw conclusions as to whether the present classifications are natural or
not. At the end we need to search for and define boundaries at subgeneric level that
Hedychieae, its taxonomical history, its structure, and its problem. The aim of the
26
CHAPTER 1: INTRODUCTION
characters) and some anatomical characters (epidermal structures and leaf transverse
section for some species represented the two subgenera, and SEM of seed coat in four
species represented the two subgenera). SEM of pollen was tried but was not successful.
The pollen were sticky one to another. I have limited time to go through the process of
carried out.
Curcuma.
27
CHAPTER 1: INTRODUCTION
Chapter Nine is a discussion of the whole aspects carried out in the study,
conclusion and suggestion for future study.
28
CHAPTER 2: PHYLOGENETIC STUDY USING
MOLECULAR DATA FROM NUCLEAR TRANSCRIBED
SPACER (RIBOSOMAL DNA) AND TRNL-F PLASTID
DNA SEQUENCES
2.1 INTRODUCTION
the nucleolar organizer (NOR) in the chromosomes (Long & Dawid 1980). rDNA
occurs in many copies, ranging from 200 copies in Linum usilassimum to 22,000 copies
(Long & Dawid 1980, Rogers & Bendich 1987). This
per haploid genome in Viciafaba
copy number varies not only between distantly related species, but also among members
of the same genus and a population of a single species (Rogers & Bendich 1987).
The many copies of rDNA exist in large arrays of tandem repeats (Figure 2.1).
The repeats consist of a gene region (pre-rRNA gene) and a spacer that separates one
gene from the next. The 5', 16-18S, 5.8S, 25S, 3' are transcribed as a single large
precursor which is processed subsequently to the mature 16-18S, 5.8S, and 25-28S RNA
molecules (Jorgensen & Cluster 1988). In some cases the repeating unit also codes for
RNA genes in eukaryotes are not linked. The
5S rRNA, but in general pre-rRNA and 5S
transcription unit of pre-rRNA has a size of 8 kb in most eukaryotes. The configuration
of ribosomal genes is usually repeated in tandem in a head-to-tail configuration. (Long
NTS is later called Intergenis Spacer (IGS). This spacer is called NTS or IGS because
regions which are transcribed into nonconserved parts of the pre-rRNA are called
transcribed spacers. These transcribed spacers are subdivided into external (ETS) and
generally highly conserved, whereas the spacer regions often exhibit extensive
29
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
intraspecific variablity in both sequence and length (Polaris el al. 1986). 1GS can vary
extensively in length among species, at the population level, or even within a single
individual (Schaal & Learn 1988 in Baldwin 1992). In contrast to the IGS, ITS is
evolutionarily conservative in length. As a result of the short length, too few restriction
sites generally occur within the ITS.
The rDNA repeat units of an individual plant are highly homogenous. This
homogeneity is presumably the result of concerted evolution of rDNA repeat units as
explained by Arnheim el al. (1980).
Several mechanisms have been thought to be the factor induced concerted
evolution such as saltatory replication hypothesis (Britten & Kohne 1968, Buongiorno-
Smith
Nardelli el al. 1972 in Li 1997), unequal crossing-over (Edelman & Gaily 1970,
1974, 1976 in Li 1997), replication slippage (Dover 1986), gene conversion (Edelman &
Gaily 1970, Birky & Skavaril 1976 in Li 1997), and duplicative transposition (Dover
1982 in Li 1997) or all called as molecular drive. The generality of concerted evolution
in multigene families have been confirmed using restriction enzyme analysis and DNA
sequencing (reviewed in Ohta 1980, Dover 1982, Arnheim 1983). Gene conversion and
unequal crossing over are probably the ultimate mechanisms for the occurrence of
concerted evolution (Li 1997). The genes evolve together through gene conversion,
unequal crossing over, and probably repeat amplification (Baldwin ci al. 1995).
30
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
31
CHAPTER 2: PHYLOGENE TIC STUDY USING MOLECULAR DATA...
32
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
Holtsford 1996), Rubiaceae (Persson 2000), and Gesneriaceae (Möller & Cronk 1997a;
Möller & Cronk 1997b; Denduangbori pant & Cronk 2000). In Zingiberaceae, it has
been used to study the classification of Curcuina (Ardiyani 1997), and the phylogeny of
Alpinia (Rangsiruji 2000), Kaeinpferia Group (Searle & 1-ledderson 2000), Roscoea
(Ngamriabsakul el al. 2000), and Hedychiuin (Wood ci al. 2000).
Molecular approach has been widely used in phylogenetic study. The product of
this study is a gene tree which hypothesises relationships among genes or genomes. The
tree resulted from this approach may not be congruent with the true species phylogeny.
This may be due to biological phenomena such as introgression, lineage sorting, and
gene duplication. In such situations, all of the nucleotides or restriction sites of a gene
or genome may be necessarily correlated as a single species tree character. The
robustness of phylogenetic hypothesis is then meaningless. However, as with other
characters, a gene tree can be combined with other characters such as non molecular
characters to be tested best by parsimony analysis (Doyle 1992).
Morphological approaches to Curcuina taxonomy continue to be significant and
probably vital. However, this cannot help to solve the problems in subgenus Curcuma.
Therefore, a molecular systematic study will be attempted. A preliminary study of the
genus has already been carried out (Ardiyani 1997). Attempts to sequence more species
in Curcuina using the ITS have been made in this study.
33
CHAPTER 2: PH YLOGENE TIC STUDY USING MOLECULAR DATA...
Figure 2.1 Repeat units of the nuclear ribosomal DNA and the organization of
the ITS region. Arrows denote orientation and approximate position of primer
sites. Primer names in quotation marks are modofied from White etal. 1990.
Primer "ITS2K" was designed by Rangsiruji (1999)
34
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
Figure 2.3 The chloroplast DNA region between the tmT (UGU) and the tmF
(GAA) genes. (i): intergenic spacer between tmT (UGU) and tmL (UAA) 5'
exon; (ii): tmL (UAA) intron; (iii): another intergenic spacer between tmL (UAA)
3' exon and tmF (GAA). The arrows with small letters indicate positions and
directions of universal primers a to f (After Taberlet etal. 1991).
35
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
2.4 MATERIALS
Wood el al. (2000), and a similar study using combined ITS and maiK sequence data by
Kress el at. (2000) show that the nearest taxa to Curcuma are Stahlianthus and Hitchenia
(not available for this study). Stahlianthus involucralus (Baker) Loes. and Smi/hatris
supraneanae W.J. Kress & K. Larsen (another related species) were therefore included
in the analysis. Two species of Roscoea (R. auriculala and R. schneideriana) and two
species of Cauileya (Ca. spicala and Ca. gracilis) were used as the outgroup to assess
the monophyly of Curcuma in respect of Stahlianthus and Smilhatris. Either their ITS
or irnL-F sequences meet the necessity for alignment. Likewise, they are distantly
enough related to enable unequivocal rooting of the tree. The sequence data of Roscoea,
and Cauileya were obtained from Ngamriabsakul el al. (2000), while those of
Siahlianlhus and Smithairis were obtained from Ngamriabsakul el al. (unpublished
data).
Ten species representing subgenus Hitcheniopsis, namely C. ecomata, C.
aurantiaca, C. australasica., C.harmandii, C. thorelii, C. alismat?folia, C. gracillima,
C. parviflora, C. roscoeana, C. petiolala, and 15 species representing subgenus
Curcuma, namely C. phaeocaulis, C. aeruginosa, C. zedoaria, C. zanihorrhiza, C.
a,narissima, C. heyneana, C. elala, C. aromalica, C. soloensis, C. cobra/a, C. longa, C.
amada, C. mangga, C. ochrorhiza, and C. purpurascens were studied in the
investigation. It is presumed that these 25 species of Curcuma represent about 25% of
species in the genus which includes almost 100 species.
Apart from the widely cultivated C. bonga, C. zanihorrhiza, C. zedoaria, and C.
aeruginosa, the rest are representative of Curcuma from Java and Thailand. It is likely
36
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
that most, if not all, of Javanese Curcuma are not wild species, but are cultivated or
escaped from cultivation and naturalized.
Verification of the species of Curcuma was accomplished by referring to the
literature on Curcuma (Roscoe 1807; Roxburgh 1812, 1832; Blume 1827; Horaninow
1862; Baker 1894; Schumann 1904; Gagnepain 1908; Valeton 1918; Holttum 1950,
(E), all other species are cultivated in the RBGE research glass house. I brought some
Javanese species from a field expedition to Java, Indonesia. Some other Thai species
the glass house were made, and when the plants were flowering, inflorescences and
flowers were collected and preserved in Copenhagen mixture (water 5.5 units; methanol
3.5 units; glycerol 0.5 units). They are deposited at E. The colours of the rhizomes and
inflorescences were matched to the Royal Horticultural Society colour chart and were
noted. Some photographs or slides were taken by myself besides those which were
taken professionally by Debbie J. White. Slides were stored in RBGE library slide
37
Table 2.1 50 accessions representing 31 taxa of used in
the molecular study (ITS and trnL-F).
Ca. gracilis (Sm.) D andy * RBGE 19820532 not known C. Ngamriabsakul 11(E) C)
Ca .spicala (Sm.) B aker* E00061739(E) E00061739 (E)
R. auriculata K.Sc h um .* RBGE 19699652 not known C. Ngamriabsakul 14 (E)
- o
R. schneideriana (Loes.) C owley * RBGK 19903345 Yunnan
St. involucratus (Baker) Loes. RBGE 19981701 Thailand C. Ngamriabsakul 34 (E)
Sm. supraneanae W. J. Kress & K. L arsen * Y. Paisooks anti vatana (BK) Thailand Y. Paisooksantivatana 00081101 (BK)
C. parvflora Wall. RBGE 19851661 Sukhothai, Thailand M. Ardiyani 31(E)
RBGE 19973659 Thailand M. Ardiyani 82(E) - o
C. thorelii I Gagnep.
C. thorelii 2 M945 Thailand M.F. Newman 945 (E)
C. roscoeana Wall. RBGE 19973658 Thailand M. Ardiyani 83 (E)
M. Ardiyani 84(E)
0
C. alismat?folia / Gagnep. RBGE 19973657 Thailand 0
C. aIismatfolia 2 M944 Thailand M.F. Newman 944 (E) m
C. gracillima Gagnep. CNG60 Phetchabun, Thailand C. Ngamriabsakul 60 (E)
00 r
C. ecoinata Craib CNG38 Chiang Mai, Thailand C. Ngamriabsakul 38 (E)
C. har,nandii Gagnep. CNG46 Chachoengsao, Thailand C. Ngamriabsakul 46 (E) C)
C. petiolata Roxb. K.M. Nagata 3688 (E) K.M. Nagata 3688 (E) C,)
C. cf. australasica Hook.f. K.M. Nagata 2312 (E) K.M. Nagata 2312 (E)
C. mangga Valeton & Zijp RBGE 19780191 Java, Indonesia M. Ardiyani 75 (E)
C. ochrorhiza I Valeton 54MA Central Java, Indonesia M. Ardiyani 54 (E)
C. ochrorrhiza 2 57MA Central Java, Indonesia M. Ardiyani 57 (E)
West Java, Indonesia M. Ardiyani 35 (BO) Cr)
C. aurantiaca / Zijp 35MA
C. aurantiaca 2 67MA East Java, Indonesia M. Ardiyani 67 (E)
M. Ardiyani 33(E)
0
C. longa I L. RBGE 19931919 not known
C. longa 2 RBGE 19782126 not known (cultivated) M. Ardiyani 85 (E)
not known (cultivated) M. Ardiyani 86(E)
0
C. longa 3 RBGE 19711837 I-
C. longa 4 60MA Central Java, Indonesia (cultivated) M. Ardiyani 60 (E) rn
(.)
C. longa 5 W81 p246 cultivated W81p246
C. longa 6 RBGE 19721701 not known (cultivated) M. Ardiyani 87(E)
0
Table 2.1 (continued) 50 accessions representing 31 taxa of used in
the molecular study (ITS and trnL-F).
MA is Marlina Ardiyani; M: Mark Newman; CNG: Chatchai Ngamriabsakul; RBGE: Royal Botanic Garden Edinburgh; RBGK: Royal
Botanic Gardens Kew; W: Waimea Arboretum and Botanical Garden, Hawaii, USA.
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
[DNA extracti on I
[Gene amplificationfPCR]
D.
r A utomated DNA sequencing
JIIIL
]
The method for total genomic DNA extraction (see Figure 2.5) is modified from
Doyle & Doyle (1987). The 2x "hot" CTAB method was used throughout. Preheated
CTAB was used instead of cold CTAB. This "hot" CTAB method is preferred (M.
40
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
Hollingsworth, pers. corn.) as it works better and faster. The method is also good for
and PVPP. The function of this extraction buffer, the 2x CTAB, is mostly to protect the
DNA from degradation by native enzymes and secondary plant metabolites. CTAB is a
cationic detergent that helps to lyse the cell membranes and will form complexes with
nucleic acids. Sodium chlorides help the formation of nucleic acid-CTAB complexes.
EDTA chelates divalent ions, especially Ca 2 and Mg2 , and prevents the activity of
metal-dependent nucleases. Beta-mercaptoethanol is a reducing agent that protects the
and quinones.
The first process is to disrupt the cell material. One circle cut out by punching
fresh healthy leaves or silica gel dried material was obtained using 1.5 ml Eppendorf
tube lid per sample. A pinch of sand was added, then all the tubes containing material
for DNA extraction were placed on a rack. In another 1 .5 ml Eppendorf tube (one tube
was needed for each DNA sample), 500 l.tl 2xCTAB was preheated with 0.2%
mercaptoethanol (1 tI) at 65 °C in a water bath. Using forceps, the tube that contains
leaf sample was submerged in liquid nitrogen (the lid of the tube must be opened or
pierced if the lid was closed). Using a small plastic pestle, the tissue (sample) inside the
tube was ground to a fine dry powder. The sample should be maintained cold by
submerging it back in liquid nitrogen. The cooling and grinding step was repeated two
to three times.
The powder obtained was dissolved in 400 j.tl preheated 2xCTAB, and then just a
pinch of PVPP was added. The tube containing the mixture was placed in a vortex for a
few seconds, then incubated for an hour at 65 °C in a water bath to allow the cell to lyse
for DNA liberation. After that, the tubes were taken out of the water bath and were
allowed to cool for 10 minutes. Then, the samples were centrifuged at 13,000 rpm
41
CHAPTER 2: PHYLOGENE TIC STUDY USING MOLECULAR DATA...
Cell disruption
mechanically by grinding, and
1
chemically with 2xC TA B
J
—01 1
1
"
-W
f pitation of nucleic acids ~Remov als
with cold isopropanol
L wit h wash b uffer
for 10 minutes at room temperature. Gently, the aqueous (upper) phase was removed
without removing any of the particulate matter, and was placed in a clean 1 .5 ml
Eppendorf tube.
400 1.11 of "wet" chloroform: i so-amyl alcohol (24:1) were added and mixed well
by inversion. The chloroform is referred to as "wet" by which addition will change the
mixtures to be slightly more hydrophilic (attracted to water). This will be able to
precipitate proteins and polysaccharides more effectively, and therefore will extract
nucleic acids better. The samples were placed on a shaker for 20 minutes for more
effectiveness instead of only inverting them by hands for a few times (M. Hollingsworth,
pers. corn.). Next, the samples were centrifuged at 13,000 rpm for 10 minutes at room
temperature. The supernatant was removed gently being careful not to pick up any of
the bottom layer, and was placed in a clean, 1.5 ml Eppendorf tube. The above steps
were repeated once again to re-extract the supernatant.
42
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
To precipitate the nucleic acids, 2/3 volume (300 .tl) freezer cold isopropanol
(also known as propan-2-ol) were added and mixed well by gentle inversion. The
mixing by inversion was continued until the oily appearance of the mixture had gone.
The samples were then left at -20 ° C overnight. After that, they were centrifuged at
13,000 rpm for 10 minutes at room temperature. At this stage, the concentration of salt
precipitated.
In the final stage, the supernatant was poured off (being careful not to pour out
the pellet), the tube was inverted and the pellet was dried in a vacuum drier for 10
minutes. It is important not to over-dry the pellet as it will stick hard to the tube and will
be difficult to dissolve it. To obtain the nucleic acid solution, the pellet was resuspended
in 50 jal of TE buffer by flicking the tube with a finger. The genomic nucleic acids can
be stored in the freezer until required.
Prior to the final stage, the pellet formed can be washed by wash buffer before it
is agitated to release the pellet from the bottom of the tube. This is to dissolve the
CTAB-nucleic acid complex and to remove the CTAB. However, throughout my study
wash buffer was not used as the DNA obtained was already clean.
thawing will induce shearing of the DNA. The final nucleic acid sample contains a
mixture of RNA, nuclear DNA, chioroplast DNA, and mitochondrial DNA.
The next step is to check the quality and quantity of the DNA obtained by
dissolved. It is best to wait until the bubbles get bigger (M. Hollingsworth, pers. corn.)
43
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
to avoid having small bubbles in the set gel. When it was cooled, I I.11 Ethidium
bromide was added. Then, it was poured to a gel mould fitted with a gel comb. When
the gel was set, the comb was removed so wells were formed.
Five .tl of extracted total genomic DNA were mixed with 3 .tl loading solution
and then loaded in a well. Five il DNA size marker (DNA 1-lyperladder) was loaded in
one side well of the samples to compare with the total genomic DNA. They were run in
an electrophoretic field at 60-80V for 1-1 .5 hours.
The negatively charged DNA will move to the positive electrode at a certain
speed which depends upon the size of the molecules. Observation of the bands of
ethidium bromide corporated-DNA was done under UV light. The results were
documented with a digital camera and printed out.
Gene amplification to produce identical DNA copies was obtained via the
Polymerase Chain Reaction (PCR) technique. Three basic stages were involved, i.e.
denaturation, annealing, and synthesis or primer extension (Figure 2.6). The
denaturation phase (high temperature) splits the double-stranded DNA into single-
stranded DNA. The annealing phase involves lowering the temperature. In this phase,
the oligonucleotide primers will bind to the single-stranded DNA. The third stage,
synthesis stage, involves the binding of polymerase enzyme (Taq= Thermus aqualicus
polymerase) to deoxyribonucleotide triphosphates (dNTP5) and catalyze a reaction by
attaching the nucleotides to the single-stranded DNA.
The ITS region was amplified using primers "ITS 5P" (5'-GGA AGG AGA AGT
CGT AAC AAG G-3') and "ITS 8P" (5'-CAC GCT TCT CCA GAG TAC A-3') (Möller
& Cronk 1997) which yielded double-stranded DNA of approximately 800 bp (Figure
2.1). The irnL (UAA) 5' exon- irnF (GAA) region (Figure 2.3) was amplified using
universal primers "c" (5'-GGA AAT CGG TAG ACG CTA CG3') and "f' (5'-ATT
TGA ACT GGT GAG ACG AG-3') (Taberlet et al. 1991). PCR amplification was
44
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
performed in the thermal cycle (GeneAmp PCR System 9600, Perkin Elmer, USA or
DNA Engine Peltier Thermal Cycler 200, Gradient Cycler, GRI).
The PCR reaction mixtures of total volume 50 tl in 0.2 ml PCR contained 34.5
p.1 sterile distilled water, 5.0 p.1 of 2 mM deoxyribonucleoside triphosphate (dNTP) mix
(Sigma Chemicals, Poole, Dorset, UK), 5.0 p.1 of lOx Bioline taqTM reaction buffer (160
mM (NH4)2SO4, 670 mM Tris HCI pH 8.8 at 25'C, 0.1% Tween-20), 2.5 p.1 of 50 mM
MgCl2, two pairs of 10 mM primers ("ITS5P" and "ITS8P" for ITS region; and "c" and
"IT for trnL-F region) each 1 .5 p.1 (Oswel DNA Service, Southampton, UK), 0.25 p.1 of
5U/p.l DynazymeTM II thermostable DNA polymerase (Finnzymes Oy, Espoo, Finland),
and 2 p.1 DNA template from aliquots of total genomic DNA. Sterile distilled water was
used instead of DNA template for the negative control. PCR cycle parameters for ITS
amplification were as follows: initial denaturation for 3 min at 94 °C; denaturation of
template DNA for 1 min at 94°C; primer annealing for I min at 55 °C; primer extension
for 1.5 min at 72 ° C. After 30 cycles, a final extension step of 5 min at 72 °C was added.
This extension was meant to allow completion of unfinished strands. PCR cycle
parameters for trnL-F region were as follow: initial denaturation for 4 min at 94 °C;
denaturation of template DNA for 0.45 min at 94 ° C; primer annealing for 0.45 min at
54° C; primer extension for 2 min at 72 ° C. A final extension step of 10 min at 72 °C was
added after 35 cycles. Gel electrophoresis (method described previously) at 60-80 V for
1-I .5 hours using 1.5 .tl of PCR products was carried out to check successful
amplification and quantity of PCR products. DNA size marker 123 bp ladder or I KB
ladder was sometimes used for comparison of amplified DNA obtained.
45
CHAPTER 2: PHYLOGENE TIC STUDY USING MOLECULAR DATA...
Isolate DNA
r cycle I
Primer extension
IIIIPII
Denature and anneal primers
Primer extension
Primer extension
Repeat cycles
Figure 2.6 General protocol of the polymerase chain reaction for amplifying
DNA (after Oste 1988 in Avise 1994)
46
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
from PCR such as primers, unincorporated nucleotides, polymerases, and salts. The
PCR products of ITS region and region between trnL (UAA) 5' exon and trnF (GAA)
were purified using the QIAquickTM PCR Purification Kit with a unique silica gel
membrane technology. The protocol from the manufacturer was as follow (see Figure
2.7).
One volume (50 p.1) of PCR products was added to five volumes (250 p.1) of
buffer PB. A QIAquickTM spin column was placed in a provided 2 ml collection tube.
The samples were then applied to the QlAquickTM column and were centrifuged at
-13,000 rpm for 30-60 sec to bind the DNA. The flow-through was discarded, and the
column was placed back into the same tube. 0.75 ml Buffer PE was added to the column
in order to wash the DNA. This was centrifuged at the same speed as before. The flow-
through was discarded, and the column was again placed back in the same tube. An
additional 1 min centrifugation was applied to the column. The next step was to place
the column in a clean 1.5 ml microfuge tube. Finally, 30 p.1 elution buffer were added to
the centre of the QlAquickTM column and it was allowed stand for I min to elute the
DNA. This was centrifuged to allow the DNA to drop down. To check the purified
PCR products, gel electrophoresis as previously described was carried out again.
sequencing PCR mixture contained: 13 p.1 of sterile distilled water, 4 p.1 of Thermo
Sequenase II reagent Premix, 1 p.1 of 5 m of one primer type, 2 p.1 of DNA template
(from purified PCR products). The samples were placed in a thermal cycler and run for
25 cycles with the following PCR conditions: denaturation step for 10 sec at 94 °C;
primer annealing for 5 sec at 50 ° C; and primer extension for 4 min at 60 °C.
47
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
PCR reaction
I-
Bind
Wash
C PO
Elute
Figure 2.7 The QiAquick spin purification procedure (modified from QiAquick
Spin Handbook)
In PCR cycle sequencing of the ITS region, two external primers identical to
those used in normal PCR, i.e. forward external primer "ITS5P" and reverse external
primer "ITS8P", were applied. In addition, two more internal primers for shorter
sequences, i.e. a reverse internal primer "ITS2K" (Rangsiruji 1999) which starts from
the far end of 5.8S, and a forward internal primer "31?" (Möller & Cronk 1997) which
starts from the beginning of 5.8S, were also employed. "ITS2K" was 5'-GGC ACA
ACT TGC GTT CAA AG-3', and "ITS31?" was 5'-GCA TCG ATG AAG AAC GTA
48
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
GC-3'. For cycle sequencing of the region between IrnL (UAA) 5' exon and trnF
(GAA), two external primers identical to those in normal PCR were employed, i.e.
forward external primer "c" and reverse external primer "f'. Two more internal primers
were also used to obtain shorter sequences, namely internal reverse primer "d" (Taberlet
el al. 199 1) and internal forward primer "e" (Taberlet el al. 1991). Primer "d" was 5'-
GGG GAT AGA GGG ACT TGA AC-3', while primer "e" was 5'-GGT TCA AGT CCC
TCT ATC CC-3'. The primers used are summarized in Table 2.2.
"ITS5P" 18S forward 5'-GGA AGG AGA AGT CGT AAC AAG G-3'
"ITS8P" 25S reverse 5'-CAC GCT TCT CCA GAC TAC A-3'
"ITS2K" 5.8S reverse 5'-GGC ACA ACT TGC GTT CAA AG-3'
"ITS3P" 5.8S forward 5'-GCA TCG ATG AAG AAC GTA GC-3'
trnL (UAA) 5' exon forward 5'-CGA AAT CGG TAG ACG CTA CG-3'
trnF (GAA) reverse 5'-ATT TGA ACT GGT GAC ACG AG-3'
P"d" trnL (UAA) 3' exon reverse 5'- GGG GAT AGA GGG ACT TGA AC-3'
trnL (UAA) 3' exon forward 5'-GGT TCA AGT CCC TCT ATC CC-3'
procedure. The 20 .tl of PCR cycle sequencing products were transferred to a fresh 0.5
ml tube containing 2 pd of sodium acetate/EDTA buffer. 55 .tl of 100% cold (-20°C)
ethanol was added to each reaction. They were mixed briefly with a vortex mixer and
were placed on ice for 15-20 min to precipitate the DNA. Then, they were centrifuged
in a microcentrifuge for 15 min at -13,000 rpm. The supernatant formed was removed
as much as possible. 250 tl of cold 70% ethanol were added to wash the pellet which
49
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
was then centrifuged at the same speed for 5 mm. The supernatant formed was again
removed as much as possible. Finally, the pellet that remained at the bottom of the tube
was vacuum-dried in a vacuum centrifuge for 2-5 mm.
Gel preparation and loading for automated DNA sequencing was performed by
M. Hollingsworth on an ABI Model 377 Prism Automatic DNA Sequencer (Perkin-
Elmer, Applied Biosystems Division, Foster City, CA, USA), in the molecular
laboratory of the Royal Botanic Garden Edinburgh, according to the manual supplied.
The sequence boundaries of each region of ITS (ITS I and ITS2) were compared
with the results of Rangsiruji etal. (2000) and Ngamriabsakul etal. (2000). Each region
was confirmed from forward and reverse sequences, for instance to verify the ITS
region, the sequence obtained from the "ITSSP" primer was compared with that obtained
from the "ITS2K" primer. The same thing was applied to the irnL (UAA) 5' exon and
trnF (GAA) comparing with the results of Rangsiruji et a! (2000). These were done
using F acturaTM version 2.0 (a program in Sequence NavigatorTM package). Another
program A utoA ssem blerTM version 2.1 (Applied Biosystems) is able to assemble the
complementary strands automatically from the four forward and reverse sequences
obtained prior to editing. A consensus sequence was then built for the whole region
using this AutoAssembler (Figure 2.8).
Sequence alignment was carried out using the CLUSTAL option in the multiple
alignment program Sequence NavigatorTM version 1.0.1 software package (Perkin
Elmer, Applied Biosystems Division, Foster City, CA, USA). These alignments were
subsequently refined by eye. The G+C content and the number and size of
insertion/deletion events (indels) were examined and determined manually. Sequence
divergence was obtained from unambiguous aligned region using PAUP* version 4.Ob4a
(Swofford 2000) in the DISTANCE MATRIX option. Other sequence characteristics
such as number of constant sites, variable sites, informative sites and autapormophic
50
CHAPTER 2: PH YLOGENE TIC STUDY USING MOLECULAR DATA...
248 250 2
AAA C ATAC AA C
AAat ATAC AA C
2K
AAA C A A C - - - AA C —
I aquem WKsisd from prism S?
1
-1211
;AOLc L\. j I
sites were calculated in PAUP* 4.Ob4a as well. The numbers of transitions and
transversions were determined using MacClade version 3.08a (Maddison & Maddison
1999).
51
CHAPTER 2: P1-I YLOGENETIC STUDY USING MOLECULAR DATA...
analysis using exhaustive search, which guarantees to find the shortest tree or trees, due
to the excessive computational time. Branch-and-bound search was applied when the
data was not too large (i.e. data of only non-polymorphic ITS sequences). This method
guarantees to find the shortest tree or trees. When the data set is large (i.e. data of
polymorphic and non-polymorphic ITS sequences), heuristic search was employed,
which does not guarantee optimality of the trees obtained but is relatively fast and
efficient.
Cladistic terminology in bold font is explained in Table 2.3.
For branch-and-bound search, MulTrees and FURTHEST addition sequence
options were selected. For heuristic search, the following strategies were applied:
SIMPLE addition sequence with TBR (Tree Bisection-Reconnection) swapping, and
RANDOM addition sequence of 500 replicates with no swapping. The resulting trees
were subjected to TBR swapping. The application of random addition sequence has
been suggested as a means to detect any multiple islands of most parsimonious trees
(Maddison 1991). The options COLLAPSE, MulTrees, STEEPEST DESCENT, and
ACCTRAN optimization were in effect.
The robustness of the phylogenetic trees was calculated using Bootstrap values
(Felsenstein 1985) and Decay indices or Bremer support (Bremer 1988, Donoghue et
al. 1992). Bootstrap analyses were performed in PAUP* 4.Ob4a with HEURISTIC
option and SIMPLE addition sequence using 1000 replicates with MAXTREE set to
1000. Decay indices or Bremer support were performed using the program Autodecay
version 4.02 (Eriksson 1998) and PAUP* 4.Ob4a. The Bremer support trees were
viewed using the program TreeViewPPC (Page 2000).
All characters are unordered and equally weighted except for a separate analysis
with characters weighted by transition/transversion ratio (MacClade). Gaps were treated
as missing data and multistate were interpreted as uncertain. A separate analysis was
conducted with coded gaps over insertion and deletion. Ambiguous regions from the
alignment, which cause alternative alignment interpretations, were excluded from the
analysis (Wojciechowski el al. 1993, Downie & Katz-Downie 1996, Möller & Cronk
52
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
1997). Analyses of ITS and trnL-F sequences were conducted separately as well as an
analysis of the combined data sets.
Descriptive statistics in the parsimony analysis were given by the Consistency
IndexCt (Kluge & farris 1969), Retention IndexRI (Farris 1989), and Resealed
Consistency IndexRC (Swofford 1993). A measure of the phylogenetic signal in the
data matrix based on skewness of a tree length distribution, called the g statistic
(Huelsenbeck 1991, Hillis & 1-luelsenbeck 1992), was made in PAUP* with 10000
random trees search options. A successive weighting approach (Farris 1969) by re-
weighting on a rescaled index was applied to reduce the effects of homoplasious
characters. This was done in PAUP* using heuristic search with TBR swapping and re-
weighted characters on Cl value.
53
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
MulTrees When selected will save all minimal trees found during branch
swapping; when not selected will save only one of the best trees
found. This option is synonymous with MulPars option in earlier
versions of PAUP.
COLLAPSE When selected will collapse any zero-length branches into polytomies
for all trees and then keep only those trees that are unique after the
collapsing is accomplished.
STEEPEST DESCENT When selected will not abandon a round of swapping until all input
trees from the previous round have been examined by the swapping
algorithm.
Decay indices or Bremer support The number of extra steps required before a dade is lost from the
strict consensus tree of near-minimum length cladograms.
rescaled consistency index=rc The product of the consistency index and the retention index of a
character.
91
Tree length distribution skewness.
54
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA..
2.6 RESULTS
2.6.1 Total genomic DNA extraction
The "[lot" CTAB method xN ith liquid nitrogen grinding method was very good
for extracting DNA in almost all the samples studied (samples either from silica gel
dried leaf material or from herbarium material). Contamination of the RNA was also
very Figure 2.9 shows the result of genomic DNA extraction from silica gel dried
leaves.
I)and'.tit' iii.b*nd
I I))))
2 3 4 5 6
111414141 1041
- SHIN) S41
IlHN) 1,)
- lUH1 50
41MM) -Il)
3114141 3(1
- 251)4) 25
204)41 20
1504) 15
- 1414)0 11141
_____ - 51141 SO
b011 (ill
44)4) 40
After comparing the total genomic DNA band with that of DNA size and
quantity marker (DNA 1-lyperladder). total genomic DNA concentration obtained was
more than 10.000 bp with approximately 15-100 ng/band or 4-20 ng/p.l varied from one
sample to another.
55
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
DNA extractions from very brown leaf herbarium material of around 17 years
old were not successful. No band came out on the gel electrophoresis. The reason for
this is probably that the DNA is degraded or there are too many inhibitors. Degradation
of DNA is probably due to the way of preserving the material, for example the use of
alcohol in preparation of herbarium specimens.
On another experiment using the same method, but applied to herbarium material
with a greenish brown colour of also 17 years old, successful results were gained.
Though the intensity of the bands was not very strong, subsequent PCR resulted in a
good DNA amplification. Unfortunately, a picture of this result (DNA extraction from
herbarium material) was not available, but PCR result from herbarium samples will be
shown later (Figure 2.12).
56
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
7
to
w I iI( I .ii e
i h1i I
4 S (i
123
fn a OR -S61
3S ,
()15
44 2
369
240
123
region -.984 bp amplicon or -.400 bp amplicon. The amplicon of -.984 bp are more
stable and are expected to be the right region. It is reasonable to use this amplicon as the
template for the next step. the sequencing step.
To obtain one single band for those species or accessions with double bands.
PCR was carried out in two separate reactions. first with primers "c" and "d", and second
with primers "e" and "1'. The combination of primers "c" and "d" resulted in region of
írnL (UAA) 5' exon to irnL ((.JAA) 3 exon. The combination of primers "e" and "1"
resulted in region of irnL (UAA) 3' exon and irnF (GAA). These methods of
57
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
I i fldI $I L
12345
—492
- (r) ()
I
a a -4OUbp
58
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA..
11234 55678
*S
Ii b Ii
50 - 0I111I,1,
hp
separating PCR in two different reactions using different sets of primers were successful
resulted in one band of approximately 350 bp. while another set of primers "3P" and
"8P" resulted in one band of approximately 600 bp (Figure 2.12).
Purification of PCR products using QlAquickTM PCR Purification Kit gave a clean result
p.180.
59
601 , I40 I20 1000 1 , 20S0, , 2I60 • 20 21201 . , 2400 , 2400,
A 1 Ci C CO C CO C F ro C C C A I Ci C F I A 1 1 AG C A T I Ci AG C A Ci CO C Ci A A A A - CI G CC CC Cl I
29 38 40 so 661
Ci C C C C CI Ci Ci C A C AG I CO CI IC Cl A AG AG CI GO Ci 1 AG C CO Ci 1 AG i CG F CG AG CA CG A iG Ci A
Be 90 1" lie 124
AC C C
RW
1160,
-
AC A
__J\
4240,
200
Ci CI A
4320,
Ci C Ci (1 AG
210
4400
C Ci Ci A
•
Figure 2.13 The electropherogram an automated DNA sequencing of ITS2 region of C. aurantiaca
showing clean non-polymorphic sequence.
.
CI A A 1 Ci
220
Base 182-190 is not shown. The height of each of the four coloured lines indicates the relative intensity of fluorescence that
corresponds to each of the four labeled dideoxynudeotides. Therefore, the peaks may be read directly as DNA sequences (bases
indicated above the electropherogram).
I C C Cl GCC AA
230
4640
CA
frJ\PvM
¶0, •
240
A Ci C
.
t\
-
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA..
The ITS regions from the two accessions of C. thorelii, C. a1isma6folia, and C.
auranhiaca are identical, so only one is shown and used for the analysis. The alignment
of ITS (Appendix 2) resulted in in a sequence 471 bp long, composed of 214 bp of ITS
and 257 bp of ITS2. The mean lengths of ITS and ITS2 of Curcuma were 193.5 and
235.5 respectively. These are slightly longer than those in Alpinia (Rangsiruji c/ al.
2000) and Roscoea ( Ngamriabsakul et al. 2000) which were 187.7 and 228.0; and
188.93 and 224.53, respectively. The sequence characteristics are summarized in Table
2.4. The alignment of irnL-F (Appendix 3) resulted in a sequence 911 bp long. The
sequence characteristics are summarized in Table 2.5.
The alignment of all taxa required insertion of 35 gaps of size Ito 4 bases, 19
gaps in ITS! and 16 in ITS2. Eighteen of these are plesiomorphies. Two gaps of one bp
size are synapomorphies uniting C. cf australasica and C. aurantiaca, while one gap
synapomorphy of two bp size unite the Thai species (C. thorelii, C. ali,cmat?fo!ia, C.
gracillima, C. parvflora, and C. harmandii). Two clades in subgenus Curcuma are
affected by a one bp sized gap synapomorphy at 472 bp grouping C. soloensis, C.
aromatica, C. elata, C. longa, C. phaeocau!is, while another one bp sized gap
synapomorphy at 486 bp groups the remaining species in the subgenus.
The alignment of all taxa required insertion of 12 gaps of size Ito 15 bases into
the trnL-F sequence. Four of these are plesiomorphies, while five are autapomorphies
for Ca. spicata (two gaps of six and one bp size), R. humeana ( one gap of five bp), C.
gracillima (one gap of one bp), and C. harmandii (one gap of 15 bp). One gap of one bp
(921 bp) is a synapomorphy of Sm. supraneae and C. roscoeana.
Pairwise comparison between the ingroups showed sequence divergence of 0-
10.55%, 0-17.33%, and 0-1 .02 for ITS I, 1T52, and trnL-F respectively. The ITS I was
less variable compared to 0-16.1% in Alpinia (Rangsiruji etal. 2000) and 0-13.86% in
Roscoea ( Ngamriabsakul etal. 2000), but the ITS2 was more variable than 0-14.6% in
Alpinia and 0-7.58% in Roscoea. The G+C content of the ITS of the species studied had
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
almost the same average as that of the Alpinia and Roscoea studies, i.e. 51.94-56.65%,
53.3-57.5%, and 5 1 .55-57.35% respectively. irnL-F contains less G+C, 32.71-33.33%.
irnL-F data. Yet, this trnL-F can be used as a support for ITS data. For example, the
consensus tree from coded gap of irnL-F data is almost congruent with that of ITS. The
subgenus Curcuma dade is separate from the Hit cheniopsis dade.
The combined data sets resulted in trees similar to those of ITS data alone
(Figure 2.18-2.19). Subgenus Curcuma forms a dade which is monophyletic supported
62
CHAPTER 2: PHYLOGENE TIC STUDY USING MOLECULAR DATA...
by BS=82 and 13I4. Two subclades (supported by BS71, 82 and Dl= 1, 2 respectively)
are detected within this dade. No morphological character corresponding to each
subc lade has been found. Subgenus Hitcheniopsis is paraphyletic, with Sm. supraneae
nested with C. ecomala or at least separated from the C. ecomata dade but nested within
the Curcuma dade. Several clades from subgenus Hiicheniopsis are well supported
such as C. peuiolata (BS89, 131=3), C. roscoeana (BS89, D13), the C. thorelii, C.
gracillima, C. alismaifolia, C. harmandii group (BS= 100, Dl= 14), and the C. cf.
ausiralasica, C. auranhiaca group (BS= 100, Dl= 10). Further study with added taxa is
needed to get a meaningful evolutionary history of these clades.
63
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA
64
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
65
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
C&.neths
- R.anñcii/uio
-
£ whjuid.:nana 0
SLinvofr,enjlus
Sm si/priw.'wae
dl?
-
d 12 (ll cf. w:OIQSILU?2
100
- Clizmniiüc.
i--- C,vI7on
Cl jjwr.'Jjj
100
100
.* Co/J4foIia
dli
Clgnwil/hm.
L C hanriindü
C:
cctioIati
C: ilinsLc
118 Cllona
d2
c:i C.pha?caiüis
dl
Claroniijell
81 .,.
-1
95 d2 -
¼'.
CQersain.oi
U14 r
Cl
7L-3 r Ca,rzula
dill
.flnuttrall vIuu (OS)
Clajm,iccthxi
IJ)
d2 C. hci'ncana
Figure 2.14 Strict consensus tree obtained from 613 equally most parsimonious trees
of length 280 steps resulting from equally weighted parsimony analysis of the combined
ITS1 and ITS2 data with gaps treated as missing data (Cl=0.7 143; Rl=0.8238;
RC=0.5884).
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
Co.gnzciiis
98 r 04
il Co- spkoJfl .;
•o
R.awiclthztIL •.D
wLII
94
R. schneidefiam. lo
SL un'oliwnz&s
Sm sup raneanae
I C. econwifa
d12 C4WAStJPrZ/ILS*WQ
dli L_ C.awwz1iaca
I C.pavflorn
C.t/wrelü
dl 100
C. au sfoLa
I I d14
FL_ c.iu
CgracUlim
61
dl
C.m3roew7lz
I C.petioloJii
C. soloen,sis
C. aionwfica
- C.ekLtlz
uI r _ C. longa
dll.
c.€&
Caentginou
Czedoajia
C. nizngga
Canwuk
15 71 C.w,uissnwz
'Decay index (Dl) di
C. heyneana
5 changes
Figure 2.15 Strict consensus tree obtained from 48 equally most parsimonious trees of
length 322 steps resulting from equally weighted parsimony analysis of the combined
ITS1 and ITS2 data with indels treated as coded present absent (C 1=0.6801;
Rk0.8147; RC0.5541).
67
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
R. hwnoa,m
C. rooeaniz
C cf. wLsbvkLs*zcgL
a peLiolvin.
• ochtorhizji
• aeiugfrwsz
• piweocaidis
• aumniiaca.
C. heyneaw
C. ltrnga
a a.nuM
97 • zedoafia
d3
• rjmthosi*izzz
C. .si!oe,ws
C. aronAzIda
C. pwpum.xens
C. cloth
C. col,f
a oJwnJifOLiii
65
dl C• FwmMzniiü
Sm sup ranew,ae
Bootstrap value (US)
C. thorelil
2 _]
'-~
A
dlDecay Index (Dl)
I charg
dl a gnicillinv
a econiii
Figure 2.16 One most parsimonius tree of length 29 steps resulting from equally
weighted parsimony analysis of the chloroplast DNA between trnL (UAA) 3 exon and
trnF (GAA) data with gaps treated as missing data (Cl=1 .000; Rl=1 .000; RC=1 .000).
68
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
£ hwna,w
- C OiWINWJOIUL
C. gmcillinz
C. econwJtz
C /wn,uwla
- C. cf aunthLsuzca
C peliolvia
C. anmJiiwa
56 i C thorelii
dl
dl dl
L _F_
— Sn sup rwteanae
C rosroeam
C ae rug flO.92
C.heyneanzz
p
C.WtthO,7*kLL
F -C. a/t)ntzjEca I
C p/W.eOca3dAS
dl
dl C. anwula e
C. iga
C. so1oen.is
C. pwpwuxens
51
dl C. co/Dram.
Bootstrap value (BS)
Figure 2.17 Strict consensus tree obtained from 55 equally most parsimonious trees of
length 53 steps resulting from equally weighted parsimony analysis of the chloroplast
DNA between trnL (UAA) 3' exon and trnF (GAA) data with indels treated as coded
present absent (Cl=0.7531; R10.7692; RC0.5793).
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
0..
4 C. econviLl
-. C.cf.wralasiaca
d9 I c:awi,iiaca
C. thorelii
76
d2
C. gnwilhinv.
100
d 14
- C. afimm4fofia
z
C. hanmnzW C,
87
dl - C.roxoetma
Cpelio/iztzz
86
d2 C.aensginoso
L.. C. zedoada
92
71
dl - C.a,mula
78
- C.heyneaiw.
d2
82
C. ochrorhi
Csoloensis
ILl
I"
21J --1
Ij 76
C. ammalka'
Decay index (DI)
--I___.
5 changes
Figure 2.18 Strict consensus tree obtained from 9 equally most parsimonious trees of
length 243 steps resulting from equally weighted parsimony analysis of the combined
ITS and chloroplast DNA between trnL (UAA) 3' exon and trnF (GAA) data with gaps
treated as missing data (Cl=0.6775; Rl=0.7265; RC=0.4922).
70
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
Ca.spicofil • OUTGROUP
Sm sup raneanae
C.econviiz
C.cfaivksaca
100
dlO
C.awrmlthca tl
C. thorelli
Cgnwilinz
100
d14
- aalism4folia
C.hwnrndii
dl C. ,toeaiuz
Cpetiolo.ta
89
C.aensguzosa
dl
C. zedoafia
89 75
d3 dl - C. ochmrhim
54
dl C. wmuhz
97 —C. heyneoiw
d5
C.longa.
CphaeocwsliN
Bootstrap value (OS)
C. salbensis
I 89
d3
\Decay Index (Dl) C. aronvika
86
Celith
3 changes
Figure 2.19 Strict consensus tree obtained from 8 equally most parsimonious trees of
length 307 steps resulting from equally weighted parsimony analysis of the combined
ITS and chloroplast DNA between trnL (UAA) 3' exon and trnF (GAA) data with indels
treated as coded present absent (Cl=0.6775; Rl=0.7265; RC=0.4922).
71
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
2.7 DISCUSSION
AND
2.7.1 PHYLOGENETIC RELATIONSHIPS WITHIN CURCUMA
BETWEEN CURCUMA AND ITS OUTGROUP
free, not connate in Smithatris, while they are adnate at the sides in Curcuma.
This genus, however, has a
Siahlianthus is closely related to Smithairis and Curcuma.
single involucre bract which is considered to be derived from two bracts joined together
(Wood el al 2000).
Analysis of the ITS region shows that C. ecomata is nested at the base in relation
to other species from subgenus Hiicheniopsis. It is nested far from the subgenus
in subgenus Curcutna,
Curcuma dade. Gagnepain (1908), however, placed C. ecomala
probably because it has a spurred anther. Analysis of the irnL-F region also shows that
dade in the analysis with coded gaps.
C. eco,na!a is nested out of the subgenus Curcuma
Gagnepain's grouping, therefore, is not supported by either molecular analysis of the
shares many other morphological characters with
ITS or the irnL-F regions. C. ecomala
the anther spurs. They share common
subgenus Hitcheniopsis despite the difference of
characters, such as the hardly developed rhizome, the free (not clasping) lateral
staminodes, and the crested anther. These are synapomorphic characters. This would
of Smithairis
made Hitcheniopsis paraphyletic. In the molecular tree (p.71), the node
and Curcuma is not resolved. A wider outgroup sampling would have enabled a more
accurate base to the limits of Curcurna.
72
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
cf ausiralasica dade. The dade is characterized by the hardly developed rhizome, the
leaves which are rounded at the base, the lateral staminodes which are not clasping, and
the ligules which are auriculate. C. auranhiaca and C. cf ausiralasica are also the only
Malesian species studied which set seeds. One character which does not unite them is
the spur on anther. C. auranhiaca has no spur, while C. cf ausiralasica has a spur on its
anther.
The existence of the C. auranhiaca and C. cf ausiralasica dade indicates that
Valeton's (1918) subgenus Paracurcuma may be a natural group. However, C.
peiiolala, which was placed in subgenus Paracurcurnaby Valeton, is not nested within
the C. auranhiaca and C. cf ausiralasica dade. Schumann (1904) placed C.
ausiralasica within subgenus Curcuma on the basis of the rhizomes which were big with
many sessile tuber. More samples of C. ausiralasica are needed for confirmation of the
rhizome character.
C. auranhiaca is distributed from Continental Asia to the Malesian archipelago,
while C. ausiralasica occurs in New Guinea and northern Australia. The dade, which is
strongly supported by the ITS data, suggests a close relationship between the two
species. C. ausiralasica may have escaped from the continental region of Asia to New
Guinea and northern Australia. The inclusion of other species of New Guinea, such as
C. meraukensis and C. laiiflora, would give more insight into the relationships among
the New Guinea species.
73
CHAPTER 2: PHYLOGENE TIC STUDY USING MOLECULAR DATA...
distributed more widely from Indo-China to Thailand (continental Asia). One species,
74
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
looking at the herbarium specimens. Therefore, they should not be assigned at specific
level. They should be treated at a lower rank in the classification.
Two subclades, well supported by molecular analysis of the ITS, are found
within the subgenus Curcuma dade. The first subclade contains C. soloensis, C.
aromalica, C. elala, C. longa and C. phaeocaulis. The second dade contains C.
aeruginosa, C. zedoaria, C. mangga, C. ochrorhiza, C. amada, C. a,narissima,and C.
irnL-F trees due to unresolved
heyneana. These subclades, however, do not exist in the
branches in many 'species'. At present there are no morphological data which support
the grouping into the two subclades. The reason for this may relate to the polymorphism
of the ITS. The polymorphic ITS sequences might have been the result of hybridization
of different sequences from different 'species'. Further study to clone the polymorphic
ITS would probably be able to verify this. However, no further study was made because
of time shortage.
The tree from molecular analysis of irnL-F does not fully support the subclades
C. ochrorhiza.
within the subgenus Curcuma dade from molecular analysis of the ITS.
C. zedoaria, and C. elata which are in one subclade of the tree resulting from molecular
analysis of the irnL-F are nested in different subclades of the tree built from molecular
analysis of the ITS. C. phaeocaulis and C. amada which are in one subclade with weak
support from the irnL-F, are also nested at different subclades of the tree resulting from
the ITS sequence data. Lack of conference probably results from different source of
DNA (nuclear versus chloroplast DNA). Analysis with more DNA regions may help to
verify this. The tree resulting from combined ITS and irnL-F data resembles that
resulting from the ITS. Use of polymorphisms of ITS is discussed in Chapter 7.
The existing sectional level classification of Curcuma is not supported either by
molecular analysis of the ITS or the trnL-F regions. Section Exaniha, those 'species'
with a lateral inflorescence, come out mixed up with section Mesantha, those 'species'
with a central inflorescence. The sectional level classification, therefore, should be
abandoned as it is only based on the position of inflorescence character which is
homoplasious.
75
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
nested between C. roscoeana and the subgenus Curcuma dade, which is also well
supported by molecular analysis of the ITS. The C. roscoeana dade supports the
intermediate characters between the two subgenera (Hitcheniopsis and Curcuma), such
as the spur on the anther which is very short (no spur or very short spur in subgenus
Hitcheniopsis vs. spurred anther in subgenus Curcuma), and the rhizomes which are
short (hardly developed rhizomes in subgenus Hitcheniopsis vs. well developed rhizome
Alpinia (ITS 1: 187-188 bp, ITS2: 226-235 bp; Rangsiruji etal. 2000) and Roscoea
(ITS 1: 188-190 bp, ITS2: 224-225 bp Ngamriabsakul et al. 2000). The length variation
76
CHAPTER 2: PHYLOGENETIC STUDY USING MOLECULAR DATA...
235-249 bp, ITS2: 196-245 bp; Möller & Cronk 1997). This contradicts the findings
from the three genera in the Zingiberaceae mentioned above. However, unlike
is almost uniform too (ITS 1: 0-10.55%, ITS2: 0.17.33% in Curcuma, ITS 1: 0-16.1%,
15.26%, ITS2: 0.41-15.57 in Aeschynanthus). This indicates the same rate of evolution
The length variation is also due to the occurrence of several indel events. The GC
contents of the trnL-F region are more or less uniform (approximately 30%) in
Curcurna, Alpinia, and Roscoea. These are much lower than those of the ITS region
(approximately 50-60%). The sequence divergence of the trnL-F region in Curcuma (0-
1.02%) is much lower than that of the ITS region (ITS 1: 0-10.55%, ITS2: 0-17.33%).
The much lower percentage of the
The same phenomena occur in Alpinia and Roscoea.
sequence divergence in the trnL-F region indicates the much slower rate of evolution of
the spacer. The tree constructed from the trnL-F sequence data is much less resolved
than that constructed from the ITS sequence data. Thus, the chloroplast spacer of trnL-F
provides very limited phylogenetic information for the present study of Curcurna.
77
CHAPTER 3: PHYLOGENETIC STUDY USING
MORPHOLOGICAL DATA
3.1 INTRODUCTION
Morphology has been used as the basis for the classification of Curcuma since
Roxburgh (1812) and the later authors, Horaninow (1862), Baker (1894), Schumann
(1904), Valeton (191 8). Inflorescence position, a character that is used for separating
sections by Roxburgh (1812), 1-loraninow (1862), Baker (1894), Schumann (1904), and
Valeton (1918), has proved to be unreliable or unstable and has been criticised by some
authors (see Chapter One). Some other characters that are used at the sectional level by
Baker (1 894) or at the subgeneric level by Schumann (1904) and Valeton (1918) are still
in question. This study aims to test whether the existing classification of Curcunia, at
the subgeneric level, reflects its evolutionary history.
Morphological study has been carried out along side a molecular study. Trees
were built from morphological data and were compared with those from molecular
sequence data. Morphological characters were also mapped onto molecular
phylogenetic trees to elucidate the possible evolutionary history of Curcuma. The
present classification of Curcuma will therefore be tested to determine whether it is
natural. The limited sampling (only around 30% of Curcuma species were examined)
makes the result of this study merely a stepping stone for a future broader study.
3.1.1.1 Rhizomes
Valeton (191 8) used rhizome characters for separating subgenus Paracurcuma,
which has a short or absent rhizome, from subgenus Curcuma. Subgenus Curcuma has
a long rhizome which forms lateral branches.
78
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
3.1.1.2 Leaves
One of several types of character that Valeton (1918) used for separating subgenus
Paracurcurnafrom subgenus Curcuma was leaf characters, i.e. base of leaves mostly
rounded, stem short, and leaves spreading. However, other species within subgenus
Hitcheniopsis (subgenus Paracurcuma sensu Valeton), such as C. alis,natfolia, have an
acuminate base and narrow lanceolate leaves. This species, however, was excluded
from the genus Curcuma by Valeton (1918).
3.1.1.3 Ligules
The ligule forms an ovate auricle, on both sides of the base of the petiole, in
subgenus Paracurcuma while it is without elongated auricle in subgenus Curcuma
(Valeton 1918). This character is easily observed from fresh material. Where
observation is not possible, data were extracted from the literature.
79
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
connected at least beyond the middle, and Hitcheniopsis having bracts which are mostly
not adnate over the middle.
3.1.1.6 Staminode
Two characters of the staminode are useful at the subgeneric level. Subgenus
Eucurcuma has longitudinally grooves and folded or clasped staminodes under the
cucullate pointed dorsal lobe, while subgenus Paracurcuma has straight staminodes and
the dorsal petal does not clasp the staminodes (Valeton 1918).
3.1.1.7 Anther
Schumann (1904) and Valeton (191 8) used anther characters in their classifications.
Schumann found spurless anthers in subgenus flitcheniopsis, and calcarate anther in
subgenus Eucurcuma. However, Schumann included C. petiolata, which in fact has a
very short spur on its anther, in his subgenus Hitcheniopsis. This received criticism
from Valeton.
Valeton made meticulous observations of anther characters to be used in his
subgeneric classification. Those are mentioned here: spur attached near the base
(Paracurcuma) versus spur attached at the back about the middle (Eucurcuma); spur not
or very shortly calcarate (Paracurcuma) versus spur calcarate (Eucurcuma); anther
grooved on the face as a continuation of the loculi (Paracurcuma) versus anther not
grooved on the face (Eucurcuma); appendix of the connective forming a short cup which
encloses the stigma entirely or its base (Paracurcuma) versus connective rounded or
narrowed towards the top, not lengthened to a cup, sometimes slightly produced between
80
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
3.1.2.2 Stigma
The stigma is unique to sectional or subgeneric level in Alpinia (Rangsiruji 1999). It
was Smith (1990) who first examined the stigmas in Alpinia and found different types of
stigmas at different levels of classification. Valeton did not observe stigma type in
Curcuma (1918).
3.1.2.3 Flower
In Streptocarpus several types of flower shape and size are found. This is connected
to different types of pollinators (Harrison et al. 1999). Different shapes and sizes of
flower in Curcurna are also observed. The morphological, anatomical and physiological
characters of flowers do not vary randomly. However, certain characteristics tend to
occur together. This is known as pollination syndromes. Red flowers are often
odourless, long-tubular, and pendant. This is the syndrome of bird pollination.
Determining the pollination syndrome of a particular flower can be a useful method of
analysing floral diversity (van der PijI 1971).
81
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
3.1.2.4 Seeds
Liao and Wu (1996) studied seed anatomy in Alpinia. So far, one type of seeds has
been found in Curcuma. However, nobody has looked at the ultrastructure of the
surface of seeds using SEM technique. This could be different among species. The
SEM of seed has been used to study the taxonomy of Cyanaslroideae (Brummit el al.
1999).
3.2 MATERIALS
3.2.1 Origin of plant materials, outgroup and ingroup taxa
Origin of plant material can be seen in Chapter Two. The rest are from
herbarium specimens which are loans from Bogor-BO, Leiden-L, and Kew-K. Living
collections (some are from rhizomes collected from field work in Java, Indonesia) are
maintained at the RBGE glass house. All but 9 accessions are vouchered at RBGE.
They will be mounted there eventually.
82
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
between any two states costs the same number of steps. Molecular data contain
midrib and edge and between apex and base. The method used partly followed Olatunji
(1980). The leaf portion was boiled in 2% sodium hydroxide solution for 5-10 minutes
in order to induce detachment of the epidermis from the other cells/tissue. Using a
scalpel, the unwanted tissue was scraped off. The epidermis tissue was then mounted on
a slide in distilled water for temporary preparation. Observation was carried out by light
microscope Axiophot Zeiss. Images were captured using Optimas 6.2. Measurements
shape and size; stomata: shape, size, type including subsidiary cells, stomatal index;
modified epidermis like hairs).
A portion of fresh leaf (as for epidermis and stomata study) was directly put in a
freezing microtome. If herbarium material was used, it was boiled in detergent solution
until it was almost back to its normal shape. Then it was put in the microtome. A tissue
of 20 pm thick was sliced on each cut. After that, it was put in a slide dropped with
stain. It was then covered by a cover glass. For permanent preparation, a solution was
dropped before covering the tissue with cover glass.
83
CHAPTER 3. PH YLOGENE TIC STUDY USING MORPHOLOGICAL DATA
3.3.2.3.2 Sputter-coating
The mounted specimens were transferred into the sputter coater chamber. Argon
was then glowed through for 10 sec. The chamber was purged to 9.3-13.3 N/rn 2 (0.07-
0.1 Torr) after the purge light came on for I mm. The specimens were coated at a preset
deposition rate (25 mA) and for a preset time (2.5 mm). The coating generates a
conductive surface, which prevents the build-up of negative charge that would interfere
in causing image distortion.
84
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
Study of the literature reveals that the small species in the study that are diploid
belong to subgenus Hitcheniopsis. Most medium sized to large species that are triploid
diploid and small habit species, and subgenus Curcuma contains mostly triploid large
species, other Curcuma species especially Indian ones, should be examined. There is a
possibility that subgenus Curcuma contains diploid and small habit species.
Does triploidy account for the large habit feature? Polyploidy can contribute to
may cause the increase in the cell size which in turn makes the whole plants to be
gigantic. Environmental factors can contribute significant change to plant size.
However, under the same environment the triploid Curcuma are bigger than the diploid
one. The genetic factor may cause the large habit on triploid Curcuma.
86
CHAPTER 3: PH YLOGENE TIC STUDY USING MORPHOLOGICAL DATA
"I
I:
A:
UI'
Figure 3.1 The habit of Curcuma.
EM
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
Character states:
0) 30to50cm
1) lto3m
and almost wholly reduced secondary rhizomes. On the other hand, subgenus Curcuma
consists of species with well-developed secondary rhizomes. The structure is shown in
Figure 3.2.
Whenever the flowering shoot springs out from the side of the rhizome, the plant will
produce a lateral inflorescence. The other way, whenever the flowering shoot comes out
from the tip, the plant will produce a central inflorescence.
The roots, that are long fihiform, spring out from the primary tuber. During the
flowering period (Valeton 1918) or by the end of the rainy season (Santapau 1945),
tubers called root-tubers form at the end of the roots. The shape may be ovate,
rhizome (primary rhizome) without any tubers (secondary rhizomes); (ii) primary
rhizomes posseses sessile tubers which are generally globose or ellipsoid; (iii) primary
88
CHAPTER 3: PH YLOGENE TIC STUDY USING MORPHOLOGICAL DATA
•:
rhizomes posseses sessile palmate (pinnate is more proper according to Valeton) tubers;
(iv) Tubers are at the end of long fibrous roots.
Compared to the rhizome architecture of other genera, such as Alpinia,
Aframomum, and Hornsiediia, Curcuma possesses a relatively simple design of
rhizomes. As a view, rhizomes of Alpinia zerumbel show architecture of nearly
hexagonal form as a primary functional requirement for exploitation of the substrate
(Bell 1979).
Why is the rhizome well developed and why it is not?
The fact that most sterile triploid species have well-developed rhizomes, and on
the other hand, fertile diploid species possess reduced rhizomes, probably indicates that
the sterility relates to the development of the rhizomes. The other theory is vice versa
where the merely vegetative reproduction has induced the ability of well-developed
rhizome propagation and induced the lost of generative reproduction. The rhizomes
serve not only as food reservoir, but also function as reproduction of the plants. Species
with reduced or undeveloped rhizomes is already successful to reproduce generatively.
Character slates:
0) Hardly developed
1) Well-develeloped
W
.
CHAPTER 3: PH YLOGENE TIC STUDY USING MORPHOLOGICAL DATA
yellow
white
bluish
91
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
Species Colour of the internal rhizome from Colour of the internal rhizome using
Valeton RHS colour chart
C. ,eUola1a Pale suiphureous -
92
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
93
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
.1
AL-
JL
I
Figure 3.4 Variation of leaf shape in an individual of C. aeruginosa
The very left, which is the oldest leaf, is elliptic while the very right,
which is the youngest leaf, is lanceolate.
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
95
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
I -•r
A
'W I
931
CHAPTER 3: PH YLOGENE TIC STUDY USING MORPHOLOGICAL DATA
'B
11
oil
Figure 3.7 Colour of sheath in Curcuma
A B
I
(A. Purple flush 2/3 length of the midrib towards the base in C. aeruginosa,
B. purple flush along the midrib in C. zanthorrtiiza. Photographed by D. White)
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
1t•
IL
,1rb
I.,
lateral spikes at the beginning of the rainy season, and central spikes at the end of the
season. He also did careful dissection and found a diminutive inflorescence from the
centre of the leaf tufts. Later on, Mangaly & Sabu (1987) reported their studies for over
8 years in South India and observed some species produce only lateral (C. zedoaria) and
some only terminal (C. longa, C. ecalcarala) and some others produce both positions. C.
amada, C. decipiens, C. inodora, C. neilgherrensis. and C. oligantha are reported by
Mangaly & Sabu (1993) as having both positions. C. amada that was described as
having central inflorescence was reported to produce only lateral ones in certain regions
(Mangaly & Sabu 1987). My observation in the research glasshouse in the Royal
Botanic Garden Edinburgh is that C. arnada produce lateral inflorescences. Santapau
(1945) was of the opinion that the classification based on inflorescence positions.
It is critical to consider that Valeton described a new species C. 'nangga for the
sole reason that it is different from C. atnada in the position of its inflorescence. He
wrote, This species is not Curcuna amada, Roxb. as I took it to be formerly (Heyne
l.c.) before I had seen the lateral scape.". C. mangga therefore could be reduced to C.
EEO
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
Imc
CHAPTER 3: PH YLOGENE TIC STUDY USING MORPHOLOGICAL DATA
Character states:
0) central
1) lateral
0) almost linear
1) elliptic or lanceolate
fertile bracts. However, in some other species, the coma can be the same colour as the
fertile bracts and shorter or as long as the fertile bracts (Figure 3.10).
This condition
referred to as having no coma, but I take it as two states, i.e. coma is shorter or as long
as fertile bracts; longer than fertile bracts.
Character stales:
Ll. .
.1
AL
F\
' 4
102
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
fertile bracts. In C. roscoeana the coma bracts are not differently coloured from the
fertile bracts. The coma and fertile bracts are orange.
Santapau (1945) reported that the colours of the coma bracts are very variable
even within a single species that he was observing in Khandala, India. The coma bract
colour of C. pseudomontana can be (i) uniform pink with various degradation; (ii) pure
white; (iii) white with pink tips, (iv) white with pink tips and a broad stripe or pink
stripes running down along the centre of the bracts; (v) white with several green stripes
running longitudinally downwards and parallel to each other; and (vi) pink with very
deep purple that is almost black (Santapau 1945). Various authors also reported at least
two variations in the colour of the coma bracts of C. longa, white or pink. The colour of
the coma bracts or the whole bracts, therefore, I think is not a reliable character defining
species.
Nakayama el al. (2000) demonstrated that malvidin 3-rutinoside is responsible
for the pigment of the coma bracts in C. alismatfolia. It was the only anthocyanin
identified from pink bracts of Curcuma alismatfolia cultivars. The concentration of
malvidin 3-rutinoside increased as the intensity of the pink colour in the bracts
increased. Does the environment affect the production of malvidin 3-rutinoside? If yes,
it could help to explain the reason why the colour of the coma bracts varies in C.
pseudomontana and C. longa.
The shape of the bracts is more stable within a species than the colour.
Environment will have less effect on this than on colour. The extreme shape is between
that of C. harmandii and other subgenus Curcuma species such as C. zedoaria, C.
zanihorrhiza, C.longa. The shape of the bracts within subgenus Curcuma is
more
uniform than that of subgenus Hitcheniopsis. Valeton was of the opinion to exclude
some species of Curcuma which should be placed in subgenus Hitcheniopsis. One of his
reasons is on the basis of the different structures of the bracts (Valeton 1918).
Character slates:
0) uniform
1) different
103
CHAPTER 3: PH YLOGENE TIC STUDY USING MORPHOLOGICAL DATA
Ifl!
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
105
CHAPTER 3: PH YLOGENE TIC STUDY USING MORPHOLOGICAL DATA
40,
IV
OP
1112.
CHAPTER 3: PHYLOGENE TIC STUDY USING MORPHOLOGICAL DATA
Character slates:
0) reddish or purplish 3) greenish
107
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
hair
Figure 3.14 Labellum shape, and hair on the middle band of the labeflurn
in Curcuma
(A. C. ecomata, B. C. gracillima, C. paiviflora, C. C. harmandii
D. C. thorelii, E. C. roscoeana, F. Curcuma subgenus Curcuma)
108
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
Character stales:
0) purple or white and purple
1) yellow or orange
boundary.
Character stales.
0) present
1) absent
me
CHAPTER 3: PH YLOGENE TIC STUDY USING MORPHOLOGICAL DATA
,
% ®r
E
0
110
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
Character stales:
0) free
1) clasping
112
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
113
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
1) funnel-shaped
nectar.
Character states.
0) absent
1) present
114
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
115
Table 3.3 Character coding for morphological data of Curcuma spp.
SPECIES
C.:anthorrhiza
C.aeruginosci
1 2
I I
I I
1
3 4 5 61 7 8 9 10 11 12 13 14 15 16 171 181 19 20 2112212314 25126 27 28 29 30 31 32 33 34 35
0 I 0 0 1 0 0 1 I I I 0 0 I 0 I I I I I I I I 3 I I I I I 1 0 I I
3 I 0 0 0 0 0 I I I I 0 0 I 0 I I I I I I I I 3 I I I I I I 0 I I
C.:edoaria 111/210000011110013111111113111111011
C.heyneana 1 1 I I 0 0 0 0 1 1 I I 1 0 0 1 3 I I I I I I I I 3 1 I I I I I 0 1 1
C.inangga I I I 2 0 0 0 0 1 I I I I 0 0 I 3 I I I I I I I I 3 1 I I I I I 0 1 1
C.phaeocaulis I I 3 I 0 0 0 I 0 I I I 1 0 0 I 0 I I I I I I I I 3 I I I I I I 0 1 1
118
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
Creston anther
21) Colour of labellum 0: present
0: purple or white and purple I: absent
1: yellow or orange
Anther dehiscence
0: along locules up to the base
22) Hair on labellum blade I: only along locules
0: present
I: absent Stigma type
0: inflated head
23) Hair on labellum band I: not inflated head
0: present
1: absent Stylar growth
0: absent
24) Shape of lateral staminodes I: present
0: linear
I: obovate to oblanceolate Ring of hair on corolla tube
0: absent
25) Arrangement of lateral staminodes I: present
0: free
I: clasping
WE
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
120
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
Ca. spc.ala
Ca. g/aPs
A. aunaiata
C.ecr,nata
C.parveiaa
i
F- C.g.raa/na
C.aJnaibJa
CMceI
V
63 C.ham&'i -
T2 C.n,scoeatia cri
- C.aurar4aca
- C.ausbalasiaca
56
- C.peicla!a
C./celga
96
- C.a'rinaica
d4
- C./eucrftza
83 - C.amada
dl C.v*iIaa
C. sbe'iss
- C.bvg
- C.cchrit#m
- C.ppu/ascens
d3
C.zeda&Ta
- C.heyneaiia V
tO
-o
C.mas7gga
Cl)
C.euct#rina
ri_
- C.9/ala
- C.amaissina
C.zan8irimiza
- C. aswgiasa
C.phaeocaui
- I change
Figure 3.19 Strict consensus tree obtained from 1000 equally most parsimonious trees
of length 75 steps resulted from equally weighted parsimony analysis of morphological
(Cl=0.5789; Rl=0.8621; RC0.4991).
121
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
the vein) and hexagonal shape (Figure 3.20). The epidermal cells are about 39-125 tm
long and 18-68 jim wide. The arrangement is in regular straight rows parallel to the
direction of the veins. Stomata consist of guard cells 30-60 gm long and 43-95 pm
wide, lateral subsidiary cells, and two or three terminal subsidiary cells. The longest
side of guard cell runs in parallel with the veins. Lateral subsidiary cells flanks the
guard cells, while terminal subsidiary cells flank both guard cells and lateral subsidiary
cells. Stomatal density is the percentage of the number of stomata to the number of
2. Stomatal density is about 1.13-2.43% in adaxial epidermis.
epidermal cells per cm
The terminal subsidiary cells run in parallel with the longest part of the epidermal cells.
Epidermal cells under the vein is smaller than those that are not under the vein (Figure
3.20).
Abaxial epidermis are mixed longitudinally elongated (the longest cell is
perpendicular to the vein), transversely elongated and isodiametric (Figure 3.21). The
epidermal cells are 17-96 gm long and 18-102 pm wide. They are randomly arranged
and are not like the adaxial epidermall cells which are in almost regular straight rows.
Stomata consist of guard cells (32-56 pm long and 35-72 pm wide), lateral subsidiary
cells, and two or three terminal subsidiary cells. The arrangement of this stomatal
complex is similar with those in adaxial. Stomata! density is 8.15-13.98% in abaxia!
epidermis. Epidermal cells under the vein is also smaller than the others which are not
mangga (Figure 3.22). The abaxial epidermal cells of C. amada are mostly transversely
elongated with some isodiametric while in C. mangga they are mostly longitudinally
elongated with some isodiametric. The arrangement is in regular straight rows in C.
nangga whereas as other Curcuma this is not so in C. amada. The abaxial epidermis
are hairy or pubescent in C. amada while they are glabrous in C. mangga. However, the
122
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
character of presence of hair is sometimes inconsistent in this case. Some C. amada can
have glabrous abaxial surface of leaves (Sirirugsa 1996). To confirm the different shape
of the epidermal cells between those of C. amada and C. mangga, more samples are
needed.
C. amarissima is quite unique in having more than two terminal subsidiary cells
(Figure 3.20-3.21). However, two terminal subsidiary cells are also found in one slide
preparation. Again, more samples are needed to confirm this.
The stomatal density is slightly lower in those from subgenus Hilcheniopsis (sampled
species are C. alismaijfolia, C. parvy'lora). Environment could affect this, but before
any conclusion is drawn, more samples should be examined. Epidermal characters are
not included in the phylogenetic analysis as morphometric analysis did not support any
groupings.
A few samples were taken for leaf transverse section investigation. From six
species, there are two types of transverse sections disregarding sectional level or even
subgeneric level. The first type has hypodermis on both sides (adaxial and abaxial), and
sclerenchyme fibres that never interrupt the lower hypodermis. The second type has
fibres in this type sometimes interrupt the lower hypodermis. The first type comprises
Figure 3.23 shows the transverse sections of some Curcunia spp. mentioned above. Leaf
transverse sections are not included in the phylogenetic analysis.
123
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
..
ra $1.i
C.
1, A. —
a/is mail/v/ia C. parviflora C. roscoeana
J -'
C. ihir/,i
-2(F 4
M .
4.. - ii
C. afnada C. :ecivaria
k!Tr
C. a,flari.s.s
ink,
f
C. I '?lc:a
'
11
C. ihr'/ii C. zedouria
Yvi
~ J_Li4J 4 Li.
¶ i'r' • 1
'
C auruflhluca
'
5() j.tni
124
CHAPTER 3: PHYLOGENE TIC STUDY USING MORPHOLOGICAL DATA
4
1
Ity
21
1
2 -
—a
- r --
00 9
_1s
C. iliorelit C. zecloaria
-I.
\-
C. aurani iaca
50 tn
125
CHAPTER 3: PH YLOGENE TIC STUDY USING MORPHOLOGICAL DATA
C. C. uniailci
5() .trn
126
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
AP
'4-
C. hevneana ( . hc iiieana
..'t. - k5'
•-1•t •'1_,/ 'e
2
ILti
C. longa C.
71
C'. rOSCOCU flU C. ro,scoeana
127
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
ridges. However, under higher magnification (2000x), these bars are not very clear, and
they turn to look like the rest of the species. The seeds of C. pebiolata, which were taken
from herbarium specimens, did not seem to dry up gradually during preservation. Hence
their surfaces are artifactual and looked rather different from the rest of the species.
128
CHAPTER 3: PH YLOGENE TIC STUDY USING MORPHOLOGICAL DATA
[ii
p-il
_
vr
129
CHAPTER 3. PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
epidermal and stomatal cells can be seen in Appendix 4. To investigate whether this is
significant, morphometric of epidermal cells and stomatal size was carried out (Table
3.4). The result shows that no groupings are found in the PCA though one cluster,
which consists of Hitcheniopsis species, seems quite separate from the rest. C.
mscoeana and C. /horelii, however, form a cluster with species from subgenus Curcurna
though rather at peripheral (Figure 3.25).
The first three components (axes) explained 66.94% of the total variance (Table
3.4). The first component, which explains 32.113% of total variance shows strong
negative correlation with width and height of adaxial and abaxial stomata, and strong
positive correlation with width of adaxial and abaxial epidermis (Figure 3.25). This
means that species with larger eigenvectors has narrower and shorter stomata and wider
epidermis. The second component explains 17.807% of total variance, and is strongly
positively correlated with width of abaxial epidermis and strongly negatively correlated
with length of abaxial epidermis (Figure 3.25). This means that species with larger
eigenvectors has wider and shorter abaxial epidermis. The third component explains
17.020% of total variance, and is strongly positively correlated with width of adaxial
epidermis and negatively correlated with width of abaxial epidermis and width of
adaxial and abaxial stomata (Figure 3.25). This means that species with larger
eigenvectors has wider adaxial epidermis, narrower abaxial epidermis, and narrower
stomata. Subgenus Hilcheniopsis generally has wider epidermis and smaller stomata.
The epidermal characters, therefore, are quite uniform within Curcuma spp. This
is probably related to the function of the epidermis, such as the stomatal for gas
exchange during respiration or photosynthesis, which is the same for all Curcuma.
130
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
% of Cum. % Broken-stick
AXIS Eigenvalue Variance Var. Eigenvalue
131
CHAPTER 3: PH YLOGENE TIC STUDY USING MORPHOLOGICAL DATA
t,,oI
S
S
*9
S
C_
• •1L.io S
..,i.,i• th6 S
• thuS
• mZ
S Ions S
eq S S S
con • S
un1
S
_4 a S •
*1*I
• j —
• *)O
S • - S
'
oon4 an.S 1on2 aue
S
sa sm
S S • —
...a•• zu*Sj
kZ • S
*4 5
rs
PWI
S
.r7 •.p.•9
S
S S
0O
•
r4 r2
S
_. — • p.,
a-S
S
AXIS!
Aw
•
•
*9
• *5
S
16
uon3 S Aruam
• •ond lO
ainarlo S
oaa7on2
S •a—S *10
P8dtfl4z.da Ion? a
• 09 .uus.j 6 # ,1
p..
a
• *9
a—s a—tO
•
S. Slo.tS •
low 0
• a—,
;rs
p.7
• Sson?
•5*4 •son) 61112
S
AXISI
Figure 3.25 Principal component plot of Curcuma.
Red dot: subgenus Hitcheniopsis, blue dot: subgenus Curcuma.
132
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
3.4.4.1 Habit
The mapping of habit characters onto the molecular tree required only one
evolutionary step (Figure 3.26). Tall habit, which is possessed by subgenus Curcuma,
seems to have evolved from small habit, subgenus Hi/cheniopsis, during the course of
evolution. The evolution of the habit is probably interfered by the evolution of
evolutionary step (Figure 3.26). Rhizome structure largely showed congruence with
combined ITS and irnL-F data. Except C. cf ausiralasica and C. petiolala, all other
species in subgenus Hitcheniopsis possess hardly developed rhizome. Well-developed
rhizome is also probably related to polyploidy, that leads to development of rhizomes.
evolutionary steps (Figure 3.26). Colour of internal rhizome has evolved several times
independently on the tree. The ancestor has whitish rhizome, which evolve to coloured
oblong leaf shape dominates the genus. Very lanceolate or rather linear leaf shape is
133
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
A Ca. grarais
B Ca grsTh
Ca c
L Ca zpica I
___
I
- 'R mc,Jaa
-
Ccamaa
Li C'gco,mta
C' cfstraiaz1a
FF
C' Cf saia,Jara
C aa,aaca
C ajr1tIaca ___________
_________• C'parv(fiora
- •Ci?1II
Ji -.C'pfiora
___
-'CthorelI,
I • C alnttfttha
L
'C' all
• C' g.aclflh.ta
L
_________
-
'Cg,'lllhna
-
'C' havmandll
ColCowta
_________________ C'ptioIia
• C'pVoh.a
C' oJtUs
C lalonit5
C Io'aga
u C' lona
L I_i =
C'ptiaucatlll
C' omtca
C' Q)ata
'I ________
C'phaocaiths
C' a,omaUca
calara
_ C' agh1osa =C aflJgrnOSa
C',*la
Czdoala
C
C'
C amlma
=
C' amaYlSShfla
H
C' ?aynQaia c'
?,ey,1kIa
C' tna_gga hadI, dt.p _________ C' matgga
— V.,'
C oc)wha — .'.I*-d.vIt C ochro,hlxa
r Ca DlltS
Ca grllll
Ca .pJCata Ca spicata
) awicuiara R aiulcijlata
C' acon,*a C' ecoraata
C' C' CfQV4salaSJtXa
C' eu,tla C .z.,ratlata
C'pftora C'parvfiora
C' ffioraill C' :horlll
C' ap.stft,lla C' alt matlftiUa
C' srac1111ma C grardlow
C' )smandll C harmaidlt
C' rosco.at C' rozcoa
C sot oqnsls
C' lottga
I_I r;== I • C lotiga
C: otnQa C arormlca
Ciata
COI .f W4fl1 ?*5fl. C 7Mgfl1Oi
C' aanagmosa
C'ata Cra&,a,la
Caa
t,,si C ,.p. at st.i. C' amgrnstma
C' h'naia C' hptaa
Ct,igga
e C oc?aerha
C'mt
C'
Figure 3.26 Mapping the habit (A), rhizome structure (B), colour of
internal rhizome (C), and shape of ligule (D) onto molecular tree.
134
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
I-,
I.)
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
Only C. harmandii has a linear bract. The rest has elliptic or lanceolate bract. The
common ancestor probably has linear bract.
Curcuma have coma bracts that are longer than the fertile bracts. Subgenus
Hitcheniopsis consists of species which has coma bracts that are longer than the fertile
bracts, and also some species which has coma bracts that are shorter than or the same
length as the fertile bracts. The short coma bracts appeared to have evolved several
times independently.
three species, C. ecomata, C. harmandii, and C. roscoeana. C. longa has two types of
colour, ie. uniform colour and different colour between, coma and fertile bracts. Except
C. longa, all other species from subgenus Curcuma have different colour between coma
and fertile bracts.
(simple type). Subgenus Hitcheniopsis possesses several type of flower, varied from
simple, complex to small type. The common ancestor appeared to have complex type.
Then it was switched to simple type in C. ausiralasica. It was switched to small type in
136
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
Curcuma. This phenomenon is most likely related to the pollination system of the taxa
step. Shape of petals is elliptic in the majority of species. The exception is C. ecomala,
The majority of the species have yellowish or oranges petal. If we re-map flower
structure onto colour of petals, we can see that except C. ecomala and C. roscoeana, all
complex and small flower have whitish or greenish petal. Simple type, in general, has
the two species, C. cf australasica and C. peliolata. The common ancestor appeared to
have dorsal petal without cucullate.
steps. The complex and small group, except C. roscoeana, have elongate labellum. On
the other hand, the complex types have roundish labellum. The common ancestor
appeared to have elongate labellum, which re-appear in complex and small type dade
137
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
steps. It is almost congruence with the mapping of the shape of labellum. The
exception is C. ecomata. Species with elongate labellum have purple or white and
purple colour, while the simple types have yellow or orange labellum. In the majority of
species with simple type, their colours of labellum are yellow or orange. The majority
evolutionary step. The majority of species have no hair on the labellum blade. C.
ecornala is the only species which has hair on the labellum blade.
evolutionary step. Hair on labellum band (Figure 3.27) is only possessed by four
species in the study, C. thorelii, C. parwflora, C. gracillima (the small type), and C.
evolutionary steps (Figure 3.27). The complex and small dade (C. thorelii, C.
gracillirna, C. alismatfolia, and C. har,nandii) has free lateral staminodes, not like the
rest of the species, which have clasping lateral staminodes. The flower of species on the
138
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
dade have little opening, while those of simple type, which have clasping lateral
staminodes, have wide opening.
139
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
this character onto molecular tree required one evolutionary step. The common ancestor
appeared to have evolved from long anther. During the course of evolution, the long
anther was replaced by short anther that fits the structure of flower.
(Figure 3.28). The mapping of this character onto molecular tree required three
evolutionary steps. The anomaly is C. ecomala, C. cf ausiralasica and C. petiolala that
have spur on the anther.
(Figure 3.28). All subgenus Curcuma have no crest on the anther, while all species
from subgenus Hitcheniopsis except C. petiolala and C. cf australasica have crest on
the anther. The outgroup has crest on the anther. The reappearance of the anther crest is
odd and interesting. Perhaps the common ancestor has a crest on the anther, and then
step. Anther dehiscence that stretches out along locules up to the base is only possessed
by C. auranhiaca.
140
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
A B Vacills
E
£vIJls 7
I c2 Ca. spIca
11 R awindara R rwuiEa
C ecoma
C cf1vaIaIaa C cfsbdhxa
L Cpirtaaca caaffarwava
p4ftoa
Ct)QIti
I - •C al1swa1ta
Ii CgriliIma
- 'C ?ms
• C mwo.a,,a
Cgracintma
C hm1II
C usco w pa
Ii [j-'Li -CoioQJ1sts
CJc'nga
Cphaociss
Csoio,,sis
C'p)kocU
Caghxsa Cr,agInoa
cz*via
C canada
Iln b.d
Cmao
.b
CC ochrorh&a CoOporhma
Ni
-Sd
c
T-
01 t.trOI ttS,.
______
_______
lICa
-
- 'C I
—C
zmz
Ica
4r:cwaa
- .CgvIi?h,oz
- .0 ?mht
- -C ,osc1a
_________________- Cpglo!o
________________________
________________
______________
_________ C oIoen.0
CphoCdfl
C 2a
C
atca
ItQ
n1gh1o0a
C n,a*a
cZ,tSSJfl
.hP'001O
Coch,t,p*bra
D
-
•
Clooga
CphoI
C
C
Camada
C
C
gz'sIs
pSCa
- 'C ail,nift,LPa
• C gTilhInoz
- •C Pa,mondn
- .0 rooco
• CpVoJata
C oIop,nl
9Ica
C augh,ow
C ochmo*fta
141
CHAPTER 3: PH YLOGENE TIC STUDY USING MORPHOLOGICAL DATA
1t!
C Ccma
C cf aAsSraiasIa?a
Li - C .Com.a
C Cf austraizIcxa
L
C a-sa L! C ajraU1a
- Cpar.sftora • Cpflora
.
.
-•CtfrwwvM,
—Cai1i,attft.JIa I' cobamill
C dtsmaa1i,a
CgraclZlhna . Cgx:flbna
- _________ • .CPdtt
__________________• C ncoeaw _________________• C ,owo
Cp1okaa
C SOIOQJ'1515
L CpV&ata
C !oensIs
C Icmga C longa
CpPocJts CphOCaU1IS
C avvmlca sC aom.!ca
C Glatig C aata
C augmosa _ C wighoa
Czw1a czedomia
Sp.n' 0 C aniada C .ai,ada
C .m,aflsshna C lssffi
Figure 3.28 Mapping spur on anther (A) and crest on anther (B) onto
molecular tree.
142
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
3.5 DISCUSSION
3.5.1 PHYLOGENETIC RELATIONSHIPS WITHIN CURCUMA
3.5.1.1 C. ecomata
C. ecomala, are nested at the basal next to the outgroups. This is supported by
the result of the analysis from molecular data. Five characters namely lateral
inflorescence, coma bracts as long as fertile bracts, hairy dorsal petal at the tip, hairy
lebellum blade, are autapomorphies of the species. The anther which is long is very
similar to that of the outgroups. As explained in Chapter Two (p.72), Gagnepain (1908)
placed C. ecomala in subgenus Curcuma. All species in subgenus Hitcheniopsis have
no spur and have central position of inflorescence. The spurred anther and the lateral
position of inflorescence are the characters that possibly encouraged him to place the
species in the subgenus Curcuma. Yet the result from morphological data does not
support his grouping either.
143
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
144
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
Curcuma. The exclusion of the species from the genus will make Curcuma a
roscoeana. Therefore, I suggest that the species in the C. parvy'lora dade are not
transferred to any other genera. More sampling of Curcuma will possibly be able to
verify this.
subclades which exist from the analysis of molecular data are not found in the tree
resulted from the morphological analysis. One subclade which contains C. elata, C.
the clasped and the grooved lateral staminodes. As it has been explained in Chapter
Two (p.75), the species in this subgenus are not good species. The result of
morphological analysis, therefore, also reveals the natural grouping of Baker's (1894)
aeruginosa and C. zanlhorrhiza), the presence of the purple flush on leaves (except in C.
also.
145
CHAPTER 3: PHYLOGENETIC STUDY USING MORPHOLOGICAL DATA
relationship of both species found in the analysis of the molecular data is separated by
the subgenus Curcuma. Therefore, the result of both the analyses differ.
The position of C. roscoeana in the tree resulted from morphological data
supports the placement of the species in Baker's (1894) section Hitcheniopsis and
supported. The position of C. petiolata in the tree resulted from morphological data
supports the placement of the species in Baker's (1894) section Hitcheniopsis,
146
CHAPTER 4: EVOLUTION OF FLORAL DIVERSITY
IN CURCUMA
4.1 INTRODUCTION
147
CHAPTER 4: EVOLUTION OF FLORAL DIVERSITY IN CURCUMA
Schum.) R.M. Sm., A. somniculosum S. Sakai & Nagam, Eleitariopsis sp., E. aff. kerbyi
R.M. Sm. were pollinated by small halictid bees (Sakai el al. 1999). The small whitish
or greenish flowers in Curcuma, such as C. parvflora, C. ihorellii, C. gracillima, C.
harmandii, maybe pollinated by small halictid bees as well. The construction of the
flowers of those Curcuina spp., such as the length of the labellum, the width of the
labellum, the length of the anther, the length of the filament, the width of the filament
and the width of the stigma, almost overlaps with that of the flowers pollinated by small
halictid bees in Sakai's study.
the flowers in the same or different pollination guilds has rarely been quantified (Sakai
el al. 1999). The correlation between flower morphology and pollination systems has
been quantitatively studied (Sakai ci al. 1999). An attempt to classify flower types that
are likely to be related to pollination syndrome has also been made (Harrison ci al.
1999).
flowers in Curcuma spp. seem to have some different patterns. To check that those
flowers have certain types, a multivariate Principal Component Analysis study was
carried out.
148
CHAPTER 4: EVOLUTION OF FLORAL DIVERSITY IN CURCUMA
4.2 MATERIALS
4.3 METHODS
attempt to classify Curcuma flowers on the basis of their quantitative variation. Twenty-
eight measurements (Table 4.2) from single flowers of 22 species of Curcuina were
taken (Appendix 5). Specimens were from fieldwork in Java (M. Ardiyani), a field
expedition to Thailand (M. Newman & C. Ngamriabsakul), and species cultivated at the
Royal Botanic Garden Edinburgh, UK. The drawing can be seen in Figure 4.1.
Measurement was carried out under the light microscope, then converted to mm by
calibration. Data were finally analysed with PC-Ord Program Version 3.18 (McCune &
Mefford 1995) with principal component analysis option. Due to limited accessions,
only five flowers of different C. longa were measured to check intraspecific variability.
Mapping of floral characters onto a molecular phylogeny can help elucidate the
evolutionary origins of floral variation within Curcurna. Floral characters were mapped
onto a molecular tree based on internal transcribed spacer (ITS) sequence data (Ardiyani
e/ at., unpubl. res. see Chapter Two). Some species included in the morphometric
analysis were omitted from the phylogeny because of unavailability of sequence data.
(Maddison & Maddison 1999). Where flower material was unavailable in those
accessions used in phylogenetic construction, different accessions were used for the
149
Table 4.1 Curcuma taxa used in this study
- Note: E is Edinburgh herbarium; BO is Herbarium Bogoriense; MA= Marlina Ardiyani; M= Mark Newman; CNG:
Chatchai Ngamriabsakul; RBGE: Royal Botanic Garden Edinburgh.
CHAPTER 4: EVOLUTION OF FLORAL DIVERSITY IN CURCUMA
152
2
47. 14
2
4 k C-
~ 27
25
26
16
15
(°:•
The eigenvalues for the first and the second axes are 17.440 and 3.592
respectively. The broken-stick eigenvalues for the first and the second axes are 3.927
and 2.927. Therefore, both the broken-stick eigenvalues are less than the eigenvalues.
If the broken-stick eigenvalue is less than the actual eigenvalue for an axis, then the axis
contains more information than expected by chance and should be considered for
interpretation (McCune & Mefford 1995). The third axis contains a broken-stick
The first two components (axes) explained 75.11% of the total variance. The
first component, which explains 62.28% of total variance (Table 4.3), shows strong
negative correlation with the overall size of the flower (Figure 4.2). This means that
flowers with larger eigenvectors have smaller overall size of flower. The largest flower
three species (C. gracillima, C. parvflora, and C. thorelii) make up a group of species
with small flowers. The rest of the species of Curcuma have larger flowers.
The second component explains 12.83% of total variance (Table 4.3), and is
positively correlated with length of the anther crest, length of the lateral staminodes at
the shortest point, length of the calyx, and length of the anther, and is negatively
correlated with width of stigma (Figure 4.2). This means that flowers with larger
eigenvectors have longer anther crests, longer lateral staminodes, longer calyces, longer
anthers, and narrower stigmas. The larger eigenvector for the second axis makes the
C. auranliaca. The type of flowers of those species is called complex. The flowers of
species which have smaller eigenvectors of the first and the second axes make a group
called simple flowers. The simple flower, therefore, has larger size, shorter anther crest,
shorter lateral staminode, shorter calyx, shorter anther, and wider stigma.
154
PCA1 PCA2
Width of stigma
Length of stigma
Width of filament at the narrowest point
Width of filament at the base
Height of filament from the insertion of lateral..
Length of anther crest C)
Thickness of anther at the widest point
To
Thickness of anther at the narrowest point
Width of anther
Length of anther (only thecae)
Length of the whole flower when flattened M
Width of labellum at the widest point 0
Width of labellum at the base
Length of labellum when flattened
Width of lateral staminode at the widest point 0
Width of lateral staminode at the base
0
71, Length of lateral staminode at the longest.. n
-
Figure 4.2 Eigenvectors of the first and the second axes for the 28 floral characteristics
CHAPTER 4: EVOLUTION OF FLORAL DIVERSITY IN CURCUMA
All three axes quite clearly define three groups of flower. As explained
previously (p. 146), the three groups are the simple, the complex and the small (Figure
4.3). The simple flower contains all the species from subgenus Curcurna studied, and
two species from subgenus Hitcheniopsis, C. cf ausiralasica and C. peliolata (Figure
4.4-4.6). The species from the subgenus Curcuina are C. !nangga, C. euchroma, C.
ainada, C. longa, C. amarissima, C. zanthorrhiza, C. purpurascens, C. aeruginosa, C.
heyneana, C. cobra/a, C. soloensis, and C. aromalica. C. ausiralasicaand C. peliolata
have flowers which are similar to those of subgenus Curcuma. Records shows that
Amegilla bees pollinate the flowers of Curcwna subgenus Curcuma (Prana 1977). The
simple flower has a very short anther crest, short and clasping lateral staminodes, shorter
anther (but quite long spur), and wider stigma. The Arnegilla bees push the spur of the
anther when they go into the simple flower. This will rotate the anther so that it touches
the back of the bee.
The small flower contains three species namely C. ihorelii, C. parvflora, and C.
gracillima (Figure 4.4-4.6). They belong to subgenus Hi/cheniopsis. They may be
pollinated by small halictid bees. The grouping of this flower type is in accordance with
the colour of the flowers which is whitish. This is in contrast with that of the simple
type which has a bright yellow flower.
(without spur), and narrower stigma. Further research to study the pollination of the
genus is needed to observe the pollinator of these species.
The position of C. pehiolala, C. purpurascens, C. aromahica, and C. aus/ralasica
are quite isolated (Figure 4.4). Their factor loadings on axis I (2.4, 3.3, 4.5 and 5.2
respectively) are between the simple and the small type. The shape is the same as those
of simple type, but the size is slightly different.
156
CHAPTER 4: EVOLUTION OF FLORAL DIVERSITY IN CURCUMA
157
CHAPTER 4: EVOLUTION OF FLORAL DIVERSITY IN CURCUMA
RO
ru. 10
0
U
N
U)
E. AV LON2 AMAD
PET
1W
Figure 4.4 Principal component plot of Curcuma. The floral types shown in Figure
43 are distinguished: the top group are complex type (A), followed by large type
( ) and the small type (•). Size and shape extremes are illustrated by the outline
drawings. Axes 1 and 2 accounted for 62.28% (Eigenvalue 17.44) and 12.83%
(Eigenvalue 3.60), respectively, of the variation.
CHAPTER 4: EVOLUTION OF FLORAL DIVERSITY IN CURCUMA
1.
0
0
U
CO)
51
Axis I score
Figure 4.5 Principal component plot of Curcuma. The floral types shown in Figure
4.3 are distinguished: the top group are complex type (A), followed by large type
(•) and the small type (•). Size and shape extremes are illustrated by the outline
drawings. Axes 1 and 3 accounted for 62.28% (Eigenvalue 17.44) and 6.73%
(Eigenvalue 1.89), respectively, of the variation.
159
CHAPTER 4: EVOLUTION OF FLORAL DIVERSITY IN CURCUMA
ROS
HAR
FR HEY
--LON1
THO
ZAN
0
U LMA/4,öUC
Cd)
AERMAN
00 pMAD
/ SOL AUR
ALl
ECO
Axis 2 score
Figure 4.6 Principal component plot of Curcuma. The floral types shown in Figure
4.3 are distinguished: complex type (A), followed by large type (•) and the small
type (•). Size and shape extremes are illustrated by the outline drawings. Axes 2
and 3 accounted for 12.83% (Eigenvalue 3.60) and and 6.73% (Eigenvalue 1.89),
respectively, of the variation.
CHAPTER 4: EVOLUTION OF FLORAL DIVERSITY IN CURCUMA
har,nandii, and C. alismaifolia also consists of two different types of flower, the
complex type (C. harmandii and C. alismahifolia) and the small type (C. thorelii, C.
parviflora, and C. gracilliina). The small type has possibly been derived from the
complex one. The dade that contains subgenus Curcurna with C. petiolaba have a
simple flower type. This type of flower has evolved twice independently. Flower type
is probably too complex to map it on to molecular tree. A character should be treated
usefully in the unitary character way of mapping it on to a molecular tree.
CHAPTER 4: EVOLUTION OF FLORAL DIVERSITY IN CURCUMA
:'a. grad/is
va. spiccita
criciktta
cor1ctta
! cfaustralasiaca
crantiaca
T'panifiora
Cthore1!i
:i a/is natifolia
C. gyacillnna
C h armandii
C roscoecrna
C.petioIata
C soIoen.sis
Clonga
CpaQocaL1is
C arornatica
Celata
C aerigmosa
C zedoaria
C arnada
C cn2arissna
C. heynecma
C rnwi gga
C ochrorhiza
162
CHAPTER 5: CHROMOSOME STUDY
5.1 INTRODUCTION
Before the study on meiosis by Ramachandran (196 1) was carried out, it was
hypothesized that the base numbers of Curcuma were 7 and 8. This was because the
first chromosome count in Curcuma (Sugiura 193 1) was 2n=64, and then Sato (1938)
reported 2n=32. It was assumed that Sugiura's plant was octoploid and Satos's was
tetraploid suggesting a base number of 8. In 1948, Raghavan & Venkatasuban reported
2n=42 in C. aromalica, so their species was hexaploid if the base number was 7.
Therefore, basic numbers of 7 and 8 were suggested for the genus. However,
Ramachandran (1961) observed regular formation of bivalents in metaphase I. This
apparent diploid (2n=42) implied that the base number is 21, and not 7 or 8.
Chromosome numbers of some Thai species (2n=32 in C. alismaifolia, 2n= 24
in C. gracillirna, 2n20 in C. harmandii, and 2n=34 and 36 in C. thorelii), assuming
they are diploid, suggest a base number that has deviated significantly. Further study is
required to understand more about chromosome number variation in Curcuma.
Existing studies of chromosome numbers in Curcuma suggest that the genus
contains diploid, triploid, and tetraploid cytotypes (Table 5.2). Cytological study of
most Javanese Curcuma species carried out by Prana (1977) reported widespread
triploidy in Javanese Curcuma. Only three species were reported to be diploid, i.e. C.
rnangga, C. auranhiaca,and C. pehiolaha. Severe sterility in Javanese species might
indicate polyploidy. However, before any conclusions are drawn, further study is
needed to check this hypothesis.
Chromosome number is one of the most widely used cytological characters in
taxonomy. This is due to the fact that in vascular plants, there is great diversity of
chromosome numbers. Chromosome number also frequently correlates with taxonomic
groupings. It often demonstrates general stability and constancy within populations,
species, and genera. This study aims to check the chromosome numbers in some species
of Curcuina from mitotic and meiotic preparations.
163
CHAPTER 5: CHROMOSOME STUDY
5.2 MATERIALS
Materials used in this study are from living collections in the Research Glass
House, The Royal Botanic Garden Edinburgh. A list of plants with their accession
number and source of origin is given in Table 5.1 below.
5.3 METHOD
The method used in this study follows Jong (1997). A flowchart of the method is
164
CHAPTER 5: CHROMOSOME STUDY
Collection of roots
Squashing
then watered regularly in the morning and late afternoon and were maintained in the
glass house at a minimum temperature of 20°C and a maximum of 30 °C. The soil was
monitored to see that it was moist enough and not too wet to suit the growth of new
roots. After one to two weeks new fresh roots were observed.
At about mid-day, healthy growing fleshy white active roots with translucent
caps were selected. They were rinsed with tap water at room temperature to clean off
any compost particles. Ten to 15 of these roots were cut with a sharp scalpel, then were
removed into a petri dish filled with distilled water. Several rinses were needed to clean
them again. A fine paintbrush helped to remove compost particles that adhered to the
roots. After that, the roots were placed on Whatman paper to blot off excess water. As
soon as possible they were immersed simultaneously into pre-treatment (or fixative) in a
sealed bottle for the required time. The bottle was given a good shake.
165
CHAPTER 5: CHROMOSOME STUDY
root tip meristem by inhibiting the formation of the spindle (Dyer 1979). Two pre-
treatment agents were tried in this study, i.e. alpha-Bromonaphthalene (aBr) and Hydro-
oxyquinolene (OQ). The roots were kept in the pre-treatment in the fridge at about 10 to
13 ° C from to six and a half hours for OQ. aBr was only tried for two hours. To allow
respiration by the roots, the bottle was shaken during this pre-treatment step to increase
oxygen in the solution. This occasional agitation also to ensured uniform penetration of
fixing fluid.
After pre-treatment, the roots were washed in two to three changes of distilled
water. Excess water was blotted off, then they were placed in fixative (Farmer's
solution) which was made from three parts of absolute alcohol (96%) and one part of
glacial acetic acid. The fixing fluid should be freshly prepared just before use. Material
fixed in an alcoholic fixative (3:1) may be stored in it or in 70% alcohol and kept in a
storage.
distilled water. After that, they were hydrolized in 5N HCI for 15 to 35 minutes at room
Feulgen staining was tried for three hours. It is a specific test for
deoxyribonucleic acid (DNA). The chemical reaction involves hydrolysis of the DNA
with colourless basic fuchsin. It gives a deep magenta colour while the other cell
components remain unstained (Jong 1996). Next, the roots were transferred to tap
water. The ions in the tap water help to fix and intensify the stain. Staining with
will help to break the cell wall while pectinase helps to break the pectin that occurs
between cells. The roots were exposed to these enzymes for 15 to 30 minutes. A
softened root was placed onto a clean slide. The root tip, which is the most densely
stained part, was cut off from the distal part and from the cap. These unnecessary parts
were removed from the slide. A few drops of 45% acetic acid were put on the root tip.
Using a blunt needle, the root tip was macerated to form fine particles. A clean
coverslip was put on top of this macerated tissue. Squashing was done by placing the
This technique is fast and satisfactory for making squash preparations permanent
with the aid of dry-ice or liquid nitrogen. First, a block of metal was frozen in liquid
nitrogen with a pair of tongs. This was then removed and placed in a polystyrene
holder. The squash preparation was placed on top of this frozen metal block with the
coverslip directly over the block for freezing. After about 30 seconds, the slide was
another coplin jar, each containing 95% or 100% ethanol. They were left for two
minutes. Both the slide and the coverslip were transferred to a second jar of alcohol and
left for two to five minutes. Next, the slide was lifted from the alcohol and was drained
on a piece of filter paper. One drop of euparal was placed near the top of material. The
coverslip was lifted and gently pushed so that the euparal was drawn towards the
material. Finally, the coverslip was lowered to cover all the material. The slide was left
167
CHAPTER 5: CHROMOSOME STUDY
was not easy. However, it was still possible to count the chromosomes in reasonably
good preparations. The chromosome numbers observed can be seen in Table 5.1. The
size of the chromosomes was not carefully measured, but the range is approximately 0.5-
1.5 Jim. Ramachandran's(196l) measurement was 0.6-1.7 Jtm. The shape of the
material (C. mangga), maintained in the glasshouse of the Royal Botanic Garden,
Edinburgh, was not doubtful. Moreover, the plant was collected and identified
previously by Prana himself. Therefore, C. rnangga has diploid and triploid forms.
and C. mangga (Prana 1977). C. aurantiaca (2n=42) was found to set seeds (Valeton
1918, Prana 1977). 1 observed this in the field. Many herbarium specimens of C.
auranhiaca have record on the seeds, however this is not the case with C. petiolala. This
is probably due to the limited member of specimens observed. C. ,nangga (2n=42,63)
was
168
CHAPTER 5: CHROMOSOME STUDY
C. heyneana 19780189 63
C. phaeocaulis 1977 1293 not successful
C. parvflora 19851661 not successful
C. mangga 19780191 63*
C. zanthorrhiza 19740965 63
C. roscoeana 19973658 not successful
C. longa 19931919 not successful
C. zedoaria 19771296 63
C. ihorelii 19973859 38?
C. alis,naiifolia 19973657 not successful
C. aeruginosa 19981 844 63
C. zanihorrhiza 197801 87 not successful
C. cobra/a 197801 88 not successful
Table 5.3 Chromosome counts from this study and from literature.
C. thorelii 34,36
38? Ardiyani (unpubl.)
170
CHAPTER 5: CHROMOSOME STUDY
reported to set seeds by Valeton ( 191 8), but neither Prana nor I observed seeds in the
field or on herbarium specimens.
Based on the study on pollen stainability and germination tests, Prana (1977)
found the diploid C. auranhiaca was fertile. C. rnangga and C. pehiolata were also
fertile, but the fertility was very low. He hypothesized that sterility in these species is
due to genetic rather than chromosomal causes. Further study is needed to verify this.
Triploidy can occur from diploid parents. When failure of reduction in meiosis
occurs, reduced and unreduced gametes (eggs or pollen) may unite. Triploidy can also
occur through hybridization between a diploid and a tetraploid. Tetraploidy in
Curcuma, however, was only recorded in C. arornahica. Nothing is known of the origin
of triploid Curcuma, whether they are formed through auto-or allopolyploidy. One
specimens collected from Java showed polymorphism in its ITS sequence (see Chapter
Seven) with one sequence resembling that of C. auranhiaca while the other was not
similar to it (data not displayed). Thus, allopolyploidy could have happened in the
species involving C. aurantiaca as one of the parents. This is in accordance with Prana's
prediction that Javanese triploid species could be hybrid forms in which C. auranhiaca
and C. inangga might have been involved. Further study focusing on those species
might be able to give insight into evolutionary history in Curcurna.
171
CHAPTER 5: CHROMOSOME STUDY
4 •1.
S • •
0
"I • .% a..
, 40
,
fl1x1ITfl
5n
Zeiss-100-1
C S
t1 t :'••
46
dip
41-
a
- ix!jn -
Mss 100-140
172
CHAPTER 5: CHROMOSOME STUDY
I t
jolt
a.
p.. I
ab so . .••
• ..7.
dQ
JI'
S's
lxlpui
I 10pm
t Zeiss-100-1 .
a
173
CHAPTER 5: CHROMOSOME STUDY
-'0
r• _
tv
at
W• %
4;
Vrb
1x1zm
lOym
Zeiss - 100- 1
a
•1
1x1pm 4t I .
10pm
Zeiss-100-1 .
4.
Figure 5.7 Chromosomes of C. zedoaria.
174
CHAPTER 6: ISOZYME VARIATION IN SOME
CURCUMA SPECIES
6.1 INTRODUCTION
175
CHAPTER 6: ISOZYME VARIATION IN SOME CURCUMA SPECIES
Allozymes
176
CHAPTER 6: ISOZYME VARIATION IN SOME CURCUMA SPECIES
chains that makes up an enzyme is required to interpret the type of pattern produced on a
gel.
The amino acids that build up enzymes have characteristic side arms that can be
positively or negatively charged, or can be uncharged or neutral. Hence, the overall net
charge of a protein will depend on the composition of amino acids that form the
polypeptide chain. Mutations in DNA sequence in genes coding for the production of
particular proteins can lead to substitutions in the amino acids that make up a
polypeptide chain. Although the molecules are still functional, the overall net charge
can be different. Amino acid substitutions may not only cause differences in the charge,
but may also alter the shape or the size of a protein. Thus, DNA sequence variation may
result in the forms of a protein that differ in net charge, size or shape. These differences
can be detected using the technique of electrophoresis.
The discovery of starch gel electrophoresis of isozymes led to studies of animals
(Harris & Hopkinson 1976), fungi (Micales 1986), plants (Soltis & Soltis 1989) and
bacteria (Selander el al. 1986). It started when Smithies (1955) described gel
electrophoresis and Hunter & Markert (1957) reported histochemical staining of enzyme
gels. Harris (1966) and Lewontin & Hubby (1966) thereafter demonstrated the simple
co-dominant Mendelian inheritance of allelic forms of isozymes, and the extent of
polymorphisms in natural populations.
177
CHAPTER 6: ISOZYME VARIATION IN SOME CURCUMA SPECIES
the nature suggested above (Wendel & Weeden 1989). Koehn & Hilbish (1987) and
Watt (1983) reported the fact of selection that can occur for specific isoforms. Ryan el
al. 1991 also reported that non-genetic variation in banding patterns could occur. This
puts doubt in the use of isozymes as population genetic and biosystematic markers.
However, careful experimental design may reduce or eliminate the potential pitfalls of
the isozyme technique. It also suggested that, when absolute measures of variation are
required, the isozyme technique may lead to under-estimates of the level of genetic
diversity. Electrophoretically detectable variation will be an underestimate of total
variation. However, the problem can be eliminated if comparative measures of variation
are required (Hollingsworth 1998).
1986; Ness ci al. 1989, Ranker & Haufler 1989, Wolf et al. 1990, Soltis et al. 1991,
Ashton & Abbot 1992, Raybould etal. 1991a, Raybould etal. 1991b, Roose & Gottlieb
1976); phylogeny (Patton & Avise 1983); variation in wild and cultivated species
(Lange & Schifino-Wittmann 2000); and taxonomic delimitation and characterization of
the germplasm (Chamberlain 1998; Lange & Schifino-Wittmann 2000).
Isozyme studies have been carried out in Curcuma to check the variation in some
species of Curcuma (Ibrahim 1996) and to identify some early flowering Curcurna
(Apavatj rut ci al. 1999). The widespread polyploids Curcuma could use isozyme
technique to investigate allo- or autoployploid origin. The formation of alloploids via
interspecific hybridization and subsequent chromosome doubling is a very important
mode of speciation in higher plants (Lewis 1980; Stace 1987). To understand the
178
CHAPTER 6: ISOZYME VARIATION IN SOME CURCUMA SPECIES
179
A flowchart of the general protocol of protein-electrophoresis can be seen in
Figure 6.2.
Dissect tissues
Electrophorese
Zymogram interpretation
180
CHAPTER 6: ISOZYME VARIATION IN SOME CURCUMA SPECIES
A vacuum hose was attached to the flask side arm, and a rubber bung was placed
on top of the flask. The flask was degassed until the small bubbles had disappeared and
large bubbles were spread evenly throughout the solution. The vacuum pump was
switched off and the rubber bung was slid slowly off the top of the flask. Then the
pattern. The gel was allowed to cool for 30 minutes. After it had set, a cling film was
wrapped over the gel before keeping it in a fridge to allow cooling to 4°C.
A washing up bowl was prepared by filling it with ice (up to 2/3 volume). A
glass beaker was filled with water and was placed in the ice bucket. A ceramic grinding
plate was put on ice. When the plate was cold (any condensation must be wiped away),
a five mm square portion (or a punched tube) of fresh and healthy leaf tissue was placed
in one of the wells. Two drops of extraction buffer were added. After that the tissue
was homogenized using a flared glass rod. As soon as the tissue was homogenized, a
filter paper wick was placed in the homogenate and was left to absorb the extract. The
above steps were repeated for the number of the samples required. A drop of food dye
was placed in an empty well and then a filter paper wick was added.
The extraction or grinding buffer was made up from LiB03 gel buffer (50m1),
KCI (37mg), M902 .6H20 (10mg), 19mg of EDTA tetrasodium salt, PVPP (25mg),
The electrode tank and sponges were washed thoroughly and dried. A sponge
was placed in each electrode tank. The blue plug was connected to the negative
(cathode), and the orange plug to the positive (anode). The tank was filled with
181
CHAPTER 6: ISOZYME VARIATION IN SOME CURCUMA SPECIES
ftc gel was removed from the refrigerator. A spatula was run around the
margins of the gel to trim any, excess from the edge of the gel former. The surface of
the gel was then covered with cling film to prevent any creases or air bubbles. On the
side of the gel former. an eight-cm running zone was marked out allowing at least five-
cm from either end of the former. The wicks are usually placed close to the cathodal
edge of the gel. The cling film was peeled back to the beginning of the running zone.
Using a ruler and the hat end of a spatula. the gel was cut through the base along the
start of the running zone. One side of the gel was pulled back gently to make a gap open
up (Figure 6.3).
Figure 6.3 Cutting and loading the gel (Murphy etal. 1996).
182
CHAPTER 6: ISOZYME VARIATION IN SOME CURCUMA SPECIES
Using a line forceps, a filter paper wick was picked up. Any excess sample
extract was dabbed off onto a paper towel. After that it was placed in the gap at the start
of the running zone. This step was repeated for the remaining samples and also for the
food dye marker. At least three-mm was allowed between wicks. After all the wicks
were in place. a wide gauge drinking straw was inserted between the short side of the gel
and the gel former end wall. The straw keeps the two parts of the gel in close contact
maximising conductivity.
i• *
ia
.-
A ll
Figure 6.4 Horizontal starch gel apparatus during electrophoresis
(Murphy et al. 1996).
The compressing force of the straw can squeeze out excess extract along the line
of wells. This was dabbed using a paper towel. The cling film was pulled back over the
wicks leaving about four cm of gel to exposed. A similar area at the opposite end was
also exposed. The gel and the gel former were placed on the electrode rig. The sponges
of each tank were placed overlapping with the exposed gel surface. The sponges must
be flat and evenly placed on the gel surface. Any excess cling film was placed at either
end of the gel on top of the sponge-gel overlap. Two A4 plastic bags were put on top of
the gel. After that, an ice pack was placed on top of the bags (Figure 6.4).
The whole rig was then placed in the fridge, connected to a power pack and run
for approximately four hours. For L1B03 buffers it was run at a constant voltage of
183
CHAPTER 6: ISOZYME VARIATION IN SOME CURCUMA SPECIES
240V which should give a current of approximately 70mA. This will decrease to
approximately 25mA after four hours. For MC8 buffers, it was run at a constant current
of 40mA, which should give a voltage of approximately 150V. After ten minutes
running, the dye should be checked to ensure that it was migrating in the correct
direction.
(40mg); NADP (7mg), MTT (12mg), PMS (3ng) in 0.5m1 of 10% M902; glucose-6-
acid (50mg); NADP (10mg), MTT (15mg), PMS (3mg) in 0.5ml of 10% MgCl2-, MgCl2
(50mg).
PGM in LiB03 was made up from Tris 0.1M pH 7.5 (50ml), glucose- I-
phosphate (100mg); NADP (10mg), MTT (15mg), PMS (3mg) in 0.5ml of 10% M902.
10% MgCl2.
184
CHAPTER 6: ISOZYME VARIATION IN SOME CURCUMA SPECIES
1DM in MC8 was made up from Tris 0.IM pH 8.0 (50ml), isocitric acid (100mg);
NADP (10mg), MTT (15mg), PMS (3mg) in 0.5ml of 10% M9 0 2: M902 (100m9).
MDH in MC8 was made up from Tris 0.1M pH 8.5 (50ml), 750mg of malic acid;
(Na salt); NAD (10mg), MTT (10mg), PMS (3mg) in ImI dH20.
GOT in LiB03 was composed of GOT substrate (SOml), fast blue BB salt
(200mg), and pyridoxal-5-phosphate (I mg). Fast blue and pyridoxal were to be weighed
dry and to be added to buffer later. GOT substrate was composed of Tris 0.IM pH 8.5
(50m1), a-ketoglutaric acid (18mg), aspartic acid (65mg), PVP 40T (250mg), 25mg of
EDTA (Na2 salt), and disodiumhydrogen phosphate (7 10mg).
SKDH in LiB03 was composed of Tris 0.1 M pH 8.0 (SOmI), shikimic acid
(60mg); NADP (10mg), MTT (15mg), PMS (3mg) in 0.5m1 of 10% MgCl2.
Tris buffers were made up from 0.IM ofTris HCI, 12.1 lgofTris Base per litre
H20. Ph was adjusted with HCI to required value.
The morpholine citrate (MC8) electrode buffer (IL) was made up from citric acid
350m1 gel buffer of morpholine citrate (MC8, I4ml of the electrode buffer was diluted in
The power pack was turned off, and left until the voltage dropped to zero. The
gel and the gel former were removed from the electrode rig. Using a spatula the gel at
the end of the running zone (around 8 cm from the wicks) was cut. The portion of gel in
front of the running zone was discarded. A notch in the top right hand corner of the gel
185
CHAPTER 6: ISOZYME VARIATION IN SOME CURCUMA SPECIES
was cut to facilitate orientation. The running zone area of the gel vas carefully lifted up
supporting it underneath and keeping it as flat as possible. After that. the running zone
portion was placed on a paper towel and the upper and lower surface was dabbed gently.
Any wicks that were still attached to the gel were removed. The gel was lifted up. and
the top surface was placed up on the gel slicer. From underneath, between the gel and
the slicer, presence of air bubbles must be checked and removed by tapping the gel
down gently. After that, a glass plate was carefully placed on top of the gel. Next, the
fishing line was held tight across the furthest end of the gel slicer from the worker. The
P PT low-
Figure 6.5 Slicing the gel for staining (Murphy etal. 1996).
The slicer, the glass plate, and the gel were inverted. Using a spatula. the thin
slice was lifted up, then was placed in the relevant staining tray. The recently exposed
surface of the remainder of the gel was dabbed with a paper towel. After that, it was
placed on the gel slicer. The previous steps were repeated for the remaining slices to get
186
CHAPTER 6: ISOZYME VARIATION IN SOME CURCUMA SPECIES
Reagents were added to gels in staining trays. The trays were covered with lids,
and they were put in an incubator in the dark at 38 °C until the bands developed. After
the bands had developed, the gel was removed to a glass plate. Finally, it was
photographed using a SLR camera.
187
CHAPTER 6: ISOZYME VARIATION IN SOME CURCUMA SPECIES
study using more samples is needed to verify this. On the other hand, PGI of the rest of
the species (i.e. C. cobra/a, C. heyneana, C. longa, C. zanthorrhiza) showed variable
patterns.
PGI of Hey I showed band types A and B. PGI of Lon 1 also produced band type
A, while that of Lon2, Lon3, Lon5, Lon 6, and Lon7 produced three bands (band type
Q. PGI of Zan l, Zan2, Zan3, and Zan4 produced three-banded phenotype of type B
while Zan2 produced slightly different band phenotype (type D). The middle and the
lowest bands in Zan2 are wider than the top band, while on the rest of C. zanihorrhiza
species the top and the middle bands are bigger than the lowest one. PGI of Co13
produced a three-banded phenotype (type F) with possible proportions of 1:2: I. PGI of
Col 1 and Co12 produced a three-banded phenotype (type B).
The patterns in PGM were also consistent with one locus with no variation,
except for Zan3 in which a two-banded phenotype was found. Therefore, C.
zanihorrhiza showed homo- and heterozygosity.
MDH is a dimeric enzyme in most plants and is involved in the oxidation of
malate to oxalacetate (Weeden & Wendel 1990). The patterns in MDH showed quite
clearly two putative loci. The fastest bands or MDH I were faint and not consistently
scorable, so they were omitted from the analysis. The slower bands or MDH2 produced
a one- to three-banded phenotype. This, however, also proved not to be readily
interpretable in terms of number of loci. Variation within taxa was only found in C.
heyneana that produced one very wide single band, and a three-banded phenotype for
the rest of the samples.
188
PGI
Aer
Amd
B
Cot
Hey
— — - - - — — - - - -_
C
Lon
D
Zan
E
Par
--
F
Col
G
Ros
PGM
Aer
Cot
Hey
B
Zan
MDH
Aer
Amd
Pha
B
Hey
Cot
Lon
C
Ros
I
D
Zed
Hey
E
Par
Amr Zan
Hey Lon Man
Lon Man Zan
00
Man Zed
Pha Zan
Zed
Figure 6.6 Isozyme phenotypes for PGI, PGM, and MDH. Letters of the alphabet designate
individual phenotypes for each isozyme. Aer= Curcuma aeruginosa, Amd C. amada, Amr= C. amarissima, Col=
C. colorata, Hey= C. heyneana, Lon= C. longa, Man= C. mangga, Par= C. paiviflora, Pha= C. phaeocaulis, Ros=
C. roscoeana, Zan= C. zanthorrhiza, Zed= C. zedoaria.
CHAPTER 6: ISOZYME VARIATION IN SOME CURCUMA SPECIES
EC
CHAPTER 6: ISOZYME VARIATION IN SOME CURCUMA SPECIES
is
CHAPTER 7: POLYMORPHISM IN ITS
7.1 INTRODUCTION
Problems with the use of nrDNA genes for molecular systematics include
1996).
Intraindividual polymorphism in ITS of most Javanese Curcuma is dealt with
here. The same phenomenon has been discovered in Winteraceae (Suh et al. 1993),
conifers (Karvonen & Savolainen 1993), peonies (Sang et al. 1995), Zea (Buckler &
Holtsford 1996), Amelanchier ( Campbell et al. 1997), Cucurbita (Jobst et al. 1998),
Gilia (Morrell & Rieseberg 1998), Castilleja (Mathews & Lavin 1998), Allium (Mes et
al. 1999), Larix and Pseudotsuga (Gernandt & Liston 1999), and Aeschynanthus
(Den duangboripant & Cronk 2000). Polymorphisms in 18s rDNA and irnK of Japanese
Here I am attempting to disentangle the two overlapping sequences by trying to find the
base insertions and deletions (indel). The method involves tracking the bases starting
from where the indel begins (Figure 7.1). Other techniques, such as DNA cloning,
would, of course, be more powerful, but this has not been possible in the time available.
192
A
C C C C G G 3 C C C C I C 3 3 3 C A C AG C 93 C
211
C C C C G T G 6 C C C T C-- 9 9 6 c a C A g t C 9 9 t C 9 a
B CG TG C C C 6 a
AIL,
T T C TC G CA C AG T C
C)
211
CCC C 3 0 F 0 C C C f c--a g 6 c a CA 9 C 9 9 C
tI
-
:ii
cc
in C. amada and C. amarissima. No species with deletion of the base is recorded yet.
194
CHAPTER 7: POLYMORPHISM IN ITS
INDELS I 11
Position (bp) 1 52 192-195
Ca. gracilis -
Ca. spicata -
R. auriculata -
R. schneideriana -
Stahlianthus sp. -
Smithatris sp. -
C. parvjflora -
C. thorelii -
C. roscoeana -
C. alismaijfolia -
C. gracillima -
C. ecomata -
C. harmandii -
C. cfaustralasica -
C. petiolata -
C. ochrorrhiza C
C. aeruginosa (-)(C)
C. amarissima (-)(C)
C. aurantiaca - TTCT
C. heyneana C
C.longal -
C. longa2 -
C. amada
C. soloensis
C. aromatica - ( ---- )(TTCT)
C. longa cf ? ( ---- )(TTCT)
C. cobra/a ( ---- )(TTCT)
C. elata ( ---- )(TTCT)
195
Table 7.2 Indel polymorphism in ITS2 region.
II III IV V VI VII VIII IX X XI
INDELS I
42-45 70-71 157- 158 170 173 174 189- 192 208-211 240-241 249-252
Position (bp) 3-5
Ca. gracilis --- ---- --
-- I - G ---- ---- --
cl
Table 7.2 (continued) Indel polymorphism in ITS2 region.
C. longa 4 ---
---- (--)(TC) ? ? ?
'.0
-.1 C. longa 5* ( --- )(TGC) ? ( ---- )(TTTA)
C. longa cf. * ( --- )(TGC) ? ? ? ? (?)(TTTA)
C. p urpurascens* ( --- )(TGC) -- ? ? ( ---- )(TTTA)
C. co lorala * ( --- )(TGC) ? ? ? ( ---- )(TTTA)
Eight Javanese species show this indel polymorphism. C. auranhiaca and C. petiolala
Analysis of each single sequence (by tracking down the indel polymorphisms in
this Chapter) shows similar result from the analysis of the consensus sequence in
Chapter Two. The tree resulted from both analysis is congruent. Further study focusing
on the indel polymorphisms of Javanese Curcuna would give more insight into the
evolutionary history of the genus. Therefore, cloning the DNA to get an isolated single
sequence is indispensable. Parsimony analyses were carried out. Different type of
analysis resulted in slightly different phylogenetic signal (Table 7.3). However, the tree
Roscoea and Cauhleya (Ngamriabsakul ci al. 2000), ITS sequence in Curcuina are found
to be polymorphic. The ITS in Curcuma shows two different modes of evolution. First,
highly homogenized copies were resulted from concerted evolution. Molecular drive
auranliaca distribute both in the continent and the Malesian archipelago. Those
homogenized-ITS species are fertile (the seeds are set). They have diploid
chromosomes. Secondly, molecular drive has been failure in the process of concerted
evolution. Length polymorphisms with indel events were found in most of Javanese and
also Ind ian polyp bid species (C. ochrorhiza, C. aeruginosa, C.phaeocaulis.
C.amarissirna, C.heyneana, C.longa, C.a,nada, C.zedoaria. C.zanihorrhiza, C..soloensis,
C.aro,naiica and C. data). This polymorphism could have been the result of incomplete
198
CHAPTER 7: POLYMORPHISM IN ITS
strict
Smithafrfr sp.
StahliuztJn,s sp.
Ca.gracilfr
Caspka1a
Rauricuiata
Rvhswidenana
C.ecimata
C.cfasstra1asiaca
C.awirutthwa
C.parviJloiva
C.thorelli
C.gnciliinw
C.ro&icoeana
C.petiolla
C. ochron*iw
C.aenighwsu a
C.aeniginosa b
C.phaeocasdLc a
C.an%LthsinMs a
C.amtzrssth,w b
C.heyneami a
C.heyneamt b
C.longalb
C.anw&sa
C.anwdab
C.zedoarial a
C.zedoarwl b
C. zedoaria2 a
C.zedoaria2b
C. zedoaria3 a
C.zedoaria3b
C.zedoarwcf. a
C.ZeJOanU cj b
C.xwthorrhiza1a
C.xantlwrrhizalb
C.xantholThiw2a
C.xautlwavhiza2b
C.soIoenis a
C.phae ocaulfr b
C.Iszgala
o
C.soloe,uis b
C.longa2a
C.Ionga2b
C.aromiUfra a
C.aronwtica b
Figure 7.2 Strict consensus tree obtained from 1000 equally most parsimonious trees
of length 167 steps resulting from equally weighted parsimony analysis of ITS2 data
of non- and polymorphic Curcuma and the outgroups, with all sites analysed
(Cl0.677; RI=0.830; RC0.562).
CHAPTER 7. POLYMORPHISM IN ITS
Strict
Srnithatm .sp
Slahlianih,ts .sp.
Ca.gnwilis
Ca..cpicuta
R.auriculgg&,
R.schn€ideria,w
C. ecoiskaigs
C. ef aistra1asiaca
C.aw,,gjaca
C.parviftora
C.ulisnwtifoligs
C. harnwuulii
C. thoreili
C.gnwiUirnrs
C.rosvoeana
C.petiolata
C. ochronhirjs
C.aenigino.ss
C.anwsis3inkI
C.hepwana
C.Iongal
C. auwda
C. zed oarial
C. zedoaria2
C. zedoa,1a3
C. zedoana cf.
C.xanthonhizgl
C.xanthorehjrj,2
C.soloensis
C.phaeocwdic
C.losaga2
C. aronwilca
Figure 7.3 Strict consensus tree obtained from 112 equally most parsimonious trees of
length 147 steps resulting from equally weighted parsimony analysis of ITS2 data of
non- and polymorphic Curcuma and the outgroups, with indel polymorphic sites
excluded (Cl=0.701; R10.835; RC=0.585).
ROU
CHAPTER 7: POLYMORPHISM IN ITS
Strict
Sinithabis .sp
Stahlianthus sp.
Ca. gncilis
Ca.spicata
&ausicsdusta
R.schnehleriana
C.econw,ta
C.cfaustralasiaca
C.awirmtiaca
C.paiviflora
C.aIisnitiJolia
C.harmmdii
C.thoslii
C. gracil1inz
C.rosvoeana
Cpetiolata
C. ochron*iza
C.anginass
C.plweocaulic
C.heyneana
C. zedoaria2
C. zedoaria3
C.xanthon*kal
C.xanthorrhka2
C.a,onKgica
C.longal
C.longa2
C.soloensis
C.Un%ZSiS3iflN$
C.ansda
C. zedoas'ial
Czedow*i cf.
Figure 7.4 Strict consensus tree obtained from 1000 equally most parsimonious trees
of length 163 steps resulting from equally weighted parsimony analysis of ITS2 data
of non- and polymorphic Curcuma and the outgroups, with indel polymorphic sites
coded as present or /and absent (Cl=0.693; R1=0.828; RC=0.574).
201
CHAPTER 7: POLYMORPHISM IN ITS
Notes: No. is number; Cl: Consistency Index; I-Il: Homoplasy Index; RI: Retention
Index; RC: Rescaled Consistency index; excl. is excluding; uninf. is uninformative.
In analysis 1, all sites were analyzed. In analysis 2, copy sequences from polymorphic
species were combined with indel polymorphic sites excluded. In analysis 3, similar to
analysis 2 but with indel polymorphic sites coded gaps as present or absent. In analysis
4, similar to analysis 3 but alignment gaps were coded as present or absent.
202a
CHAPTER 8: REVISION OF JAVANESE CURCUMA
203
CHAPTER 8: REVISION OF JAVANESE CURCUMA
lecto. excl. descr. since the description of Linnaeus does not describe C. longa. Burtt in
1981 proposed again Curcuma Roxburgh with C. longa as a type. But this is rejected by
the Committee and Curcuma Linnaeus is at the end conserved for the generic name of
the commercial turmeric. The history is summarized and tabulated in Table 8.3.
(Appendix 8) are from Royal Botanic Garden Herbarium Edinburgh (E), and loans from
Herbarium Bogoriense (BO), Leiden Herbarium (L), and Kew Botanic Gardens
Herbarium (K).
Data on species name, authority, collector, collection number, date of collection,
collection site, latitude, longitude, etc are stored in Pandora Data Base in the Royal
Botanic Garden Edinburgh. Descriptions were written by hand instead of with the Delta
204
CHAPTER 8: REVISION OF JAVANESE CURCUMA
1 737 Linnaeus in Musa Cliffortiana included only Curcuma rotunda in genus Curcuma.
1753 Linnaeus in Genera Plantarum added C. longa without modifying the description of the
genus, _so_the _description _was _only _based on C. rotunda.
Curcuma rotunda was transferred to other genus Boesenbergia, therefore C. longa is the
only species which can be chosen as lectotype of Curcuma so long as the identity is clear
1918 Valeton in his study of Zingiberaceae of Java and Malaya rejected C. longa as nomen
dubium on the basis of that the original description and illustration of Hermann referred
to C. aromatica Salisb. He proposed C. domeslica Valeton as the correct name for C.
longa L. This was followed by, for example Trimen (1931), Holttum (1950), Backer&
Bakhuizen f. 1968).
1923 Britton & Wilson chose C. longa as a lectotype of the genus.
1959 Mansfeld pointed out ifthe typification of C. longa proposed by Merrill is accepted, C.
longa_can_be_taken_as_lectotype_of the name Curcuma.
1972 Burtt & Smith proposed to conserve Curcuma Roxburgh (1810) non Linnaeus (1753)
with C. angustifolia Roxb. as its type species. Their reason was that there was no
support of the choice from Britton & Wilson (1923) and Hitchcock & Green (1929).
However, if the identity of C. longa L. as valid species can be established, it does not
preclude the re-adoption of C. longa.
1974 The Committee rejected Burtt & Smith's proposal to conserve Curcuma Roxb. They
agreed with Mansfeld (1959) so regarded C. longa as a lectotype.
1977 Burtt reinvestigated C. longa and found out Manjella Kua of Rheede can be chosen as
lectotype of Curcurna.
1981 Burtt revised the proposal and proposed again to conserve Curcuma Roxb. on the basis
of that Roxburgh gives a description of C. longa while there is no description in
Linnaeus (1753) which can cause the genus only be cited Curcuma L. quoad lecto. excl.
descr.
1984 The Committee agrees with Burtt in saying that the lectotypification of C. longa is in
conflict with the protologue. However, committee voted 10-I (one abstention) that the
conserved name should date from 1753 and be attributed to Linnaeus. Curcuma
Linnaeus is conserved with a new type C. longa Linnaeus.
205
CHAPTER 8: REVISION OF JAVANESE CURCUMA
8.3 RESULTS
8.3.1 Generic re-description
Curcuma L.
Plant of about 30-155 cm height. Rhizome white, bright yellow, yellow orange, deep
orange internally, pungent or young mango-like fragrant, 3.0-11.0 by 1.5-2.5 cm. Leaf-
sheath green, glabrescent or pubescent, edge sometimes hairy, c. 5.0 to more than 40.0
cm long. Leafless sheath green or reddish brown, broad linear, round, mucronate at apex,
glabrescent to pubescent, sometimes hairy on its edge, the outer one smaller than the
inner one, c. 4.0-35.0 cm long. Petiole green, c. 0-22.0 cm long, glabrous or glabrescent.
Liguie c. 1.0-3.0 mm by 1.2 to 3.0 cm, ciliate, auriculate or not. Blade green with or
without purplish brown flush on distal half of the leaves on upper or on both surface,
lanceolate to broadly lanceolate, acuminate at apex, rounded, slightly acuminate, acute to
obtuse decurrent at base, glabrous or glabrous and hairy on edge at tip on upper surface,
glabrous or glabrous and hairy on edge at apex on one side on lower surface, c. 1 6.5-85.0
by 5.0-23.0 cm. Midrib green, dark red brown on upper surface, green on lower surface.
Inflorescence central or lateral. Scape green, c. 6.5-50.0 cm long, slightly glabrescent or
pubescent or puberulous, covered with 3-6 leafless sheaths. Spike c. 10.0-20.0 by 4.0-9.0
cm. Leafless sheath on scape c. 4.0-24.0 cm long, glabrescent or pubescent to densely
pubescent. Spike c. 9.0-19.0 by 3.5-9.0 cm. Fertile bract green tipped with purplish, c.
8-20, oblong, orbicular, obovate, broad ovate, broad elliptic to rounded, rather acute or
obtuse to slightly rounded, glabrescent or pubescent to densely pubescent on outer
surface, pubescent on inner surface, c. 3.5-6.5 by 1 .6-5.2 cm. Coma bract purple at tips,
dark green getting dull white towards base, c. 4-9, lanceolate to broad lanceolate to broad
elliptic, acute to slightly obtuse or obtuse to rounded, slightly mucronate or mucronate or
not so, pubescent to densely pubescent on outer surface, c. 3.5-8.4 by 0.9-4.5 cm.
Bracleole boat-shaped, c. 1.6-3.5 by 0.9-2.6 cm, sparsely shortly hairy with dense hair at
apex, white pelucid or white with pink top. Calyx c. 1.0-1.4 cm long and c. 1.35 cm
wide, three-toothed, with one the deepest among others, hairy especially at tips, apex of
tooth truncate slightly cleft, white pelucid and pinkish at tips. Corolla tube c. 1.6-3.2 cm
long, white, pale yellow, pink, yellowish white at the base, pinkish at apex. Corolla
lobes pink, pale pink, reddish or brownish. Dorsal corolla-lobe elliptic-rounded, ovate,
hooded with cucullate apex, c. 1.0-2.0 by 0.6-1.4 cm. Lateral corolla-lobes broad
oblong, elliptic, apex rounded, c. 1.0-1.5 by 0.7-I .1 cm. Labellu,n almost orbicular with
blunt or pointed apex, shallowly cleft at apex, c. 1.4-2.0 by 1.2-1.8 cm, and c. 1.0-1.2 cm
wide at base, citrine, yellow, light orange, orange, median darker with purple spot in the
centre. Lateral siamninodes unequal elliptic or oblong with slightly rounded apex, broad
Wel
CHAPTER 8: REVISION OF JAVANESE CURCUMA
obovate, c. 1.0- 1 .6 cm long and c. 6.0-9.0 mm wide at base, c. 8.0-18 mm at the widest
part, light yellow, light orange, orange. An/her slim, c. 3.5-4.0 mm long, c. 2.0-2.5 mm
thick, thecae c. 1.5-2.0 mm wide, protruded on posterior side, spur almost as long as
theca, c. 2.0-4.0 mm long, curved. Filament c. 3.5-8.0 by 4 mm. Style thin and slender.
Stigma bilabiate with saccate of two-lobes on dorsal side. Sty/odes 5.0-7.0 mm long.
Ecology: teak forests, dry grassy lands, cultivated, waste ground and abandoned
cultivation.
Altitude range: 0 - 2500 m
Rhizome developed; leaves narrowed at the base; spurs are almost as long or half as
long as the anther; lateral staminodes folded; ligule not auriculate..........................
.................................................... subgenus Curcuma
Rhizome hardly developed; leaves rounded at the base; no spurs or very short spurs on
anther; lateral staminodes not folded; ligule auriculate ......... subgenus Hitcizeniopsis
The key leads involve poor characters such as colours and smells. They are best applied
to living material.
Rhizomes bitter, white with blue tinge; leaves with purple flush ..........................2
Rhizomes light yellow, yellow, dark yellow or orange; leaves green or with purple
fl u sh................................................................................................ 3
Rhizome bitter, not mango smell; leaves with intense purple streak along the midrib..
.................................................. . zedoaria
Rhizome slightly bitter, mango smell; leaves green..........................................5
Rhizome with clear mango smell, very slightly bitter; inflorescence lateral.............
.......................................... mangga
Rhizome very slightly manggo smell, bitter; inflorescence central........................
................................................. ochrorlziza
207
CHAPTER 8: REVISION OF JAVANESE CURCUMA
Inflorescence all green or coma bracts pinkish; rhizome dark orange, turmeric smell...
longa
Inflorescence all greens; rhizome orange .......................................... viridiflora
Rhizome dark orange or yellow-orange; coma bracts pinkish, flower bracts greenish
..................... . . .
13
Rhizome dark yellow; bracts all green....................................... purpurascens
Finding a type citation or citing a type for Curcunia longa was beyond the scope of the
thesis.
Rhizome deep orange (21A or 22A) externally and internally, the young tips white. Blade
green. Inflorescence central. Flower bract green. Coma bract white or white with
purple towards. Flower pale yellow. Corolla tube white. Corolla lobes white. Lateral
corolla-lobes ovate, rounded at apex. Labelluimi creamy white with yellow median band.
La/eral siamninodes creamy white.
208
CHAPTER 8: REVISION OF JAVANESE CURCUMA
Vernacular. Kunyit
Altitude range:
250- 1281 m
Rhizome cylindric, bluish inside, bitter. Blade green with purplish brown flush on distal
half of the leaves on upper surface (on both surface, Holttum), midrib green.
Inflorescence lateral. Flower bract green tipped with purplish. Coma bract purple at
tips, dark green getting dull white towards base. Calyx transparent and pinkish at tips.
Corolla tube yellowish white (dark pink red, Valeton; deep-crimson pink, Holttum).
Corolla lobes reddish or brownish (pale red, Baker). Lateral slaminodes light yellow.
Vernacular. Koneng hideung, Kunyir hideung (Sunda), Temu ireng (Jawa), Temo
ereng (Madura).
Note. aeruginosa is meant for the aeruginous colour of the rhizomes. The specimen
cited in the protologue comes from Pegu. Roxburgh reduces Rumph's "temu itam"
(which is C. aeruginosa Roxb.) to a Bengalese species called C. caesia Roxb.
Rhizome yellow (20C) in the centre, orangish (25D) and bluish in the middle, and white
or cream at peripheral; bitter. Leafless-sheath reddish brown. Leat,sheath reddish brown
with green at apex. Petiole green. Blade green with purple streak along the midrib;
midrib green. Inflorescence lateral. Flower bract elliptic, acute, green tipped with
purplish. Coma bract red purple at top and white at the lower half. Corolla lobes red.
Labellum deep yellow.
Note. The description on inflorescence is taken from Valeton 1918. In daily use, the
rhizomes of this species is mixed up with those of C. aeruginosa.
Altitude range:
177-301 m
209
CHAPTER 8: REVISION OF JAVANESE CURCUMA
Curcuma longa L. var. zedoaria (Christm.) Ardiyani Monandr. Pt. Scitam. 1828:
41
Rhizome cylindric, light yellow internally (20C). Leafless-shea/h green with brown flush,
mucronate at apex. Leaf-sheath green. Petiole green. Blade green with reddish brown
flush along the midrib on both surface, midrib purplish green on upper surface, green on
lower surface. Inflorescence lateral. Scape green. Flower bract elliptic (broad obovate,
Horaninow), green tipped with purplish. Coma bract dark pink at tip getting dull white
towards the base. Calyx yellowish white with pinkish at tip. Corolla tube yellowish
white. Corolla lobes white. Labelluni yellow, median darker with purple spot in the
centre. Lateral staminodes light yellow.
Altitude range:
50 - 626 m
Curcuma longa L. var. mangga (Valeton & Zijp) Ardiyani in Bull. Jard. Bot.
Buitenz. XXVI. 1918: 50
Rhizome young mango-like fragrant, light yellow inside (3C when young; 20B on
peripheral and 25B in thecentre when old) (citrine, Valeton). Leqfless-sheath green.
Leaf-sheath green (sometimes slightly purple, Holttum). Petiole green. Blade green,
midrib green. Inflorescence lateral. Scape green. Flower bract oblong to elliptic, obtuse
to rather acute, green tipped with purplish. Coma bract dark pink at tips getting dull
white towards base. Calyx pelucid. Corolla tube yellowish white at the base, pinkish at
apex. Corolla lobes pinkish white (flower pure white, Valeton, Holttum). Lateral
siaminodes light yellow.
Vernacular. Ternu mangga; Tema, Tema poh (Madura, East Java and Yogyakarta);
Temu bajangan (local name in Bojonegoro), Temu lalab (Jakarta).
Note. mnangga means mango. The rhizome has young mango-like odour.
210
CHAPTER 8: REVISION OF JAVANESE CURCUMA
Altitude range:
250 - 259 m
Rhizome lightyellow (I B) when old and yellowish white (3C) when young. Blade green.
Inflorescence central. Flower bract broad oblong ovate, obtuse, light green. Coma bract
obovate, obtuse to slightly acute, white with rose apex. Corolla tube pale yellow. Corolla
lobes pale rose.
Note. Specimen cited in the protologue i.e. Heyne 705 from Randublatung. ochrorhiza
is named from the externally and internally white, in the centre greenish-lemon tinged
rhizome (Valeton 1918).
Altitude range:
259 - 260 m
Rhizome nearly pure yellow mixed with brown tinged (deep yellow, Roxburgh)
internally. Root tuber ovate, very light ash-coloured with gold yellow endodermis.
Blade green, ,nidrib very faintly purplish on upper surface (Holttum). Inflorescence
central. Flower bract narrowly ovate, obtuse, light green. Coma bract snow white with
partly light brown dots, sometimes with sporadic light brown dots at apex. Corolla lobes
faintly pink, the rest of the flower light cream.
Vernacular. Temu lati, Lati putih, Temu kebo, Temu prit (Jawa).
Note. A native of Sumatra, and other eastern islands. Roxburgh described the species
from a specimen from Bencoolen (Bengkulu) which was sent by Dr. Charles Campbell
(Roxburgh 1820). Specimen cited in the protologue is a specimen of Dr. Charles
Campbell from Bencoolen (Bengkulu). viridiflora probably means green flower or
inflorescence. The description above is from Valeton (1918).
Altitude range:
138 -600m
211
CHAPTER 8: REVISION OF JAVANESE CURCUMA
Curcuma longa L. var. heyneana (Valeton & Zijp) Ardiyani in Bull. Jard. Bot.
Buitenz. XXVI. 1918: 54;
Rhizome yellow (7A) inside (pure bright yellow, Valeton; whitish sometimes yellowish
in the centre, Backer & Bakhuizen). Leqf-sheath green. Petiole green. Blade green,
midrib green. Inflorescence lateral. Scape green. Flower bract green tipped with
purplish. Coma bract dark pink at tip getting dull white towards base. Calyx transparent
and pinkish at tip. Corolla tube yellowish white at the base and pinkish at apex.
Corolla lobes pinkish white.
Vernacular. Temu giring (Central Java), Jaha (West Java), Temu giring, Tema licin,
Tema koneng (East Java), Tema lateng (local name in Mt. Yang).
Altitude range:
177 -900 rn
Rhizome pure lemon yelow internally, yellowish white externally. Root tuber very pale
yellowish internally. Inflorescence central. Scape pubescent. Flo%I'er bract pale yellow
green on lower bracts, pale green with violet stripes on upper bracts, pubescent. Coma
bract almost white at the base, red violet upperhalf towards the apex, pubescent. Corolla
lobes very light pink.
Note. Specimen cited in the protologue i.e. specimens from Randublatung. The
description above is based on the herbarium specimen from Randublatung with Valeton's
handwriting. The data on underground parts is adopted from Valeton's protologue.
Rhizome orange internally (25A), bitter and minty fragrance. Root tuber ellipsoidal, light
grey with yellow or lemon yellow endodermis. Blade green. Jnflorescence central.
Flower bract very light pure green on lower bracts, with spotted violet at apex on upper
bracts (Valeton). Coma bract white with pink towards the apex, dark violet at apex.
Corolla lobes very light.
212
CHAPTER 8: REVISION OF JAVANESE CURCUMA
Note. Specimen cited in the protologue, i.e. Heyne 50 from Solo. soloensis from the
word Solo, a town in Central Java.
Altitude range:
50-610 m
Rhizome orange inside (21A or 22A). Root tuber orange (10E) when young, orange
(I 7A or B) when old. Lea/less-shea/h green with brown flush. Leaf-sheath green.
Petiole green. Blade green with narrow purplish brown flush along the midrib on both
surface, purple flush remain on down the middle in old leaves, midrib purplish green on
upper surface. green on lower surface. In/lorescence lateral. Scape green. Flower bract
broadly ovate, rather acute or pointed, green tipped with purplish. Coma bract dark pink
at tip getting dull white towards base. Calyx transparent or colourless (toothiets light red,
Valeton). Corolla tube yellowish white. Corolla lobes pinkish white. Lateral
.s'taminodes light yellow with pinkish spot at the back or whitish according to Holttum.
Vernacular. Temu lawak (Central/East Java), Koneng gede (Sunda), Temu labak
(Madura).
Note. The species that was described by Roxburgh came from Amboyna (Ambon) which
was brought to the Botanic Garden at Calcutta in 1798. However, this plant flowered for
the first time in April and May 18 10 (Roxburgh 1820).
Altitude range:
177 -400m
Enum.pl.Javae (1827)46
Rhizome yellow (I 7C). Root tuber elliptical, orange yellow in the inner cortex, grey
pleroma. Blade green, midrib dark red brown on upper surface. Inflorescence central.
Flower bract light green. Coma bract white at the base, light green to nearly white
towards the apex, light brown spotted at apex. Bracteole pellucid white. Corolla lobes
snow white, the rest of the flower very pale cream yellow. Lateral stamninodes elliptic,
falcate, obtuse.
-... 13
L
CHAPTER 8: REVISION OF JAVANESE CURCUMA
Note. Specimen cited in the protologue is from province of Bantam (Banten), West Java.
The word puipurascens is probably for the purple flush on the leaves. The description of
flower is based on the herbarium specimen.
Altitude range:
260 - 850 m
Rhizome bright orange (25A or B) internally, the young one bright orange yellow
internally. Root tuber orange yellow. Blade green, midrib reddish on upper surface,
almost disappear on mature plant. Ii?florescence central. Flower bract broad ovate,
green with red purple tip. Bracteole pelucid and pink at apex. Coma bract light green at
base red purple at apex. Flower diluted ochraceous. Corolla lobes pink (56A). Corolla
tube yellow. Lateral staminodes light orange. Labelluin light orange with darker
(orange) median band. Filament orangish. Anther and spur white.
Vernacular. Kunir batok, Temu prit, Temu lati.
Note. Specimen cited in the protologue, ie. Heyne 449 from Mojokerto; Heyne 52 from
Kediri; Heyne s.n. from Soemenep (Sumenep) Madura. euchroma means well-coloured.
Altitude range:
50 - 308 m
Rhizome deep orange (25B) internally, smell pleasant, taste mild or carrot-like
(Holttum). Root tuber orange in the centre, grey on edge. Blade green, midrib
dark red
brown on both surfaces. Inflorescence central. Flower bract green on lower bracts, green
with violet stripes and pink at top on upper bracts, pubescent. Coma bract white or light
green, dark purple at apex, pubescent. Corolla lobes pale pink (Valeton).
Note. Specimen cited in the protologue, ie. Heyne 35; Backer 11348 from Mount Willis;
K.1645 from Randublatung. cobra/a is probably meant for the colour of the coma
which is dark purple, and the flower which is orange (Valeton 1918). The tallest flowers
1918).
of any Curcuina of Java. It has some resemblance to C. peiiolata(Valeton
214
CHAPTER 8: REVISION OF JAVANESE CURCUMA
Altitude range:
177 -600 m
Rhizome hardly developed, orange (21 A or 22A) internally. Blade green. Inflorescence
central. Flower bract green. Coma bract pink (55B or Q. Flower pale yellow. Corolla
tube orange (28B). Corolla lobes orange (28B). Lateral corolla-lobes ovate, rounded at
apex. Lahellum orange (28B). Lateral staminodes orange (28B). Anther whitish with no
spur.
Altitude range:
50 - 550 m
Rhizome hardly developed, very pale sulphurous internally. Blade green. Inflorescence
central. Flower bract green, dark purple-brown at tip. Coma bract dark purple-brown.
Flower very light orange. Corolla tube white. Corolla lobes white with a yellow or pink
top. Lateral corolla-lobes rotundate-ovate-oblong. Labellum light orange with yellow
median band. Lateral siaminodes light orange. Anther with short spur. (Valeton 1918)
Note. petiolata means long petiole. In Java, the species is cultivated in Batavia and
Buitenzorg and seems to be rare (Valeton 1918). The species is native from Pegu and
Martaban. It was first discovered by Mr. F. Carey which then sent to the Calcutta
Botanic Gardens. Then, it was described by Roxburgh (Hooker f. 1870). The species is
closely related to the turmeric (C. longa) and C. australasica Hook. f. The two latter
species, however, have narrow leaf at the base, longer spikes, and not too deep pouches
of its bracts (Hooker f. 1870).
Altitude: 100
215
CHAPTER 8: REVISION OF JAVANESE CURCUMA
oil
iien leaf sheath of Rhizome well-developed
ihuenus C urcuma -
'
ro
Rhizome section white
I No%Nn teal lieath of . with blue tinged
•
huenus ( 'ureuma
4
Ligule auriculate Rhizome section light
i '4 !L yellow
r
Rhizome section orange -
Leaf green, midrib brown. colour chart designed
ithout purple flush
NN
216
CHAPTER 8: REVISION OF JAVANESE CURCUMA
Inflorescence lateral -
bracts pinkish, flower
bracts ireemsli
L Lateral inflorescence
C Central inflorescence
217
CHAPTER 8: REVISION OF JAVANESE CURCUMA
aura ntiaca
petiolata
L At
-.
i
-
-- -
'- longa
aeruginosa
C brog
33
fig
riH -
J
_-
fr colorata
euchmma
L - .. heyneana
.- -
L
iiT'
pow
1- mangga
ochrorhiza
C
4* phaeoca u/is
q
- I -Alt
K7 purpurascens
pp,
Ic p soloensis
pp,
viridiflora
zanthorrhiza
zedoana
I
Figure 8.2 Illustration of Javanese Curcuma.
From the left to the right: inflorescence, rhizome section, rhizome structure, leaf, ligule, and
leaf sheath. An eye with a slash indicates the material is not seen;.C: central; L: lateral.
218
CHAPTER 9: GENERAL DISCUSSION
AND CONCLUSIONS
The tree resulting from the combined molecular and morphological data can be
seen in Figure 9.1. It is less resolved than those resulting from the molecular or the
morphological data. The subgenus Curcuma dade is nested terminal in the tree. This is
supported by the analysis of the molecular or the morphological data. Therefore, the
present classification of the genus into the subgenus Curcuma is confirmed. Two
subclades within the subgenus Curcuma dade are similar with those in the molecular
tree. However, this does not reflect the two sections in the subgenus. The sectional
level classification is not supported, and should be abandoned. As mentioned in Chapter
Two, there are no morphological data which support the grouping into the two
subclades. C. petiolata is nested next to subgenus Curcuma dade. This is supported by
the analysis of the molecular or the morphological data. Hence, the species is closely
related to the subgenus Curcuma having an intermediate character between the two
subgenera in Curcurna. The C. auranhiaca and C. cf australasica dade, the C.
parvflora dade, C. roscoeana, and C. ecomata are nested in a polytomy. Nevertheless,
the data show the close relationship among species of the subgenus Hitcheniopsis.
Therefore, subgenera Curcuma and Hiicheniopsis are phylogenetically distinct. The C.
parviflora dade is well resolved. C. harmandii, which has a complex floral type, is
nested at the base of the dade. C. thorelii and C. gracillima, which have a small floral
type, are nested at the terminal end of the dade. It appears that the floral type has
evolved from the complex to the small type.
219
Ca
afkaft Ca go*
- - A a*zh
Caor
Cw
4-
4-
SKAwnnrMw
CAMM
-
A(",- -
I
C,rad
CAMN C"NUM I
1 r44 r Cp.*3
c
4-- - - -
-Cho
II
cvvxovt c- 4-
CbP
C- 1
C.ih Ic C-
r
cimm
flrL.1 L Cz
CF
CJLVW C2
lc.- Caeyara
Cnwge
C*a
CRdMM
Cqp
catra —Cai
Ccth r C&
Ic'
-Ji —1 me
Figure 9.1 Trees constructed from molecular data (the left), morphological data (the
middle), and combined both molecular and morphological data (the right)
CHAPTER 9: GENERAL DISCUSSION AND CONCLUSIONS
with well-developed rhizomes set seeds. This sterility may result from constant
vegetative reproduction using the rhizomes which have been intensively used as
traditional medicines. During the course of evolution, the rhizomes of Curcuina may
have evolved from none or very short to well developed rhizomes. The process had
from the subgenus Hitcheniopsis, however, has narrow leaves acuminate at the base.
This character, therefore, is not consistent with the division of the two subgenera. The
ligules are auriculate for all species from subgenus Hiicheniopsis. They are not
auriculate in subgenus Curcuma. So far, this ligule character is consistent with the
according to Valeton (1918). They are connected at least partly beyond the middle in
a good for separating the two subgenera. The coma bracts in subgenus Hiicheniopsis are
221
CHAPTER 9: GENERAL DISCUSSION AND CONCLUSIONS
shorter than the fertile bracts (Valeton). C. a1ismatfolia has shorter fertile bracts than
coma bracts. This character of the length of the coma bracts is therefore inconsistent
the dorsal corolla lobe in subgenus Curcuma (Valeton 1918). These characters are
congruent with the division of the subgenera. Free (not folded) and non-grooved lateral
to enter the flower, it has to push the spurs. This in turn will rotate the anther, since the
anther is versatile, and will position it so that it touches the back of the pollinator. The
pollen then will be trapped on the pollinator's back and will be carried to other flowers.
The connectives of the anther cells lengthen at the apex to form a crest which
encloses the stigma entirely or only its base. This anther crest occurs in all species from
222
CHAPTER 9: GENERAL DISCUSSION AND CONCLUSIONS
Paracurcuma does not accommodate the rest of the species from the subgenus
inaccurate. They are the shape of the base of the leaves, the adnation of the bracts, the
length of the coma bracts in comparison with that of the fertile bracts, and the spurs on
the anther (p.221-222). Some other characters are good for the delimitation of the
subgenera, i.e. the shape of the ligules, the groove on the lateral staminodes, the clasping
the inflorescence has been proved homoplasious. The shape of the apex of the bracts in
subgenera. Although the delimitation of the subgeneric taxa is not really appropriate,
(p.75; p.145).
The classification of Curcuma by Velayudhan etal. (1996) is mainly based on
the rhizome characters. Due to limited samples, the classification can not be completely
checked. In the field, C. !onga (which is in the section Tuberosa subsection two) was
found to produce flower spikes from the tip of the sessile tuber, and from the tip of the
primary mother rhizome in another clone. Therefore, the division into subsections in
section Tuberosa does not seem natural. Further study is needed to verify this.
223
CHAPTER 9: GENERAL DISCUSSION AND CONCLUSIONS
both sides of the base of the petiole. Lateral staminodes longitudinally grooved, folded
under the dorsal lobe. The connective of the anther cells not lengthened towards the top,
C. longa L., C. longa var. aeruginosa, C. longa var. zedoaria, C. longa var. amada, C.
longa var. heyneana, C. longa var. ochrorhiza, C. ion go var. soloensis, C. longa var.
arornatica, C. longa var. elata, C. longa var. amarissima, C. longa var. zanthorrhiza, C.
longa var. brog, C. longa var. euchroma, C. longa var. cobra/a, C. longa var. mangga,
C. longa var. ochrorhiza, C. longa var. purpurascens, C. longa var. viridflora, and C.
longa var. phaeocauiis.
both sides of the base of the petiole. Lateral staminodes not longitudinally grooved, not
folded under the dorsal lobe. The connective of the anther cells lengthen towards the top
forming a crest.
Further study that includes more samples of Curcuma sp. is needed to check if the
224
APPENDICES
prior to use.
U TE (10mM Tris-HCI and 1 mM EDTA).
U IOxTBE buffer stock (89mM Tris-HCI, 89 mM boric acid, 2mM EDTA pH 8.0).
225
APPENDIX 2. Sequence data matrix (displayed from 5' to 3') of aligned ITS1 and ITS2 regions of 28 taxa of
Zingiberaceae. Numbers in bold italic (1 to 35) indicate the number and position of alignment gaps. Uncertain nucleotide
states are coded based on PAUP conventions (Swofford 1993) as follows: K=G/T, M=A/C, R=A/G, S=C/G, W=AIT, Y=C/T,
N=AII7GIC. Square brackets at the end of sequences show the real spacer length of ITS1 plus lTS2 regions.
30 40 50 60 70 80 90
10 20
. 1
ITS1 . . . . . . .
. 0
1234 . . . . 5 .6789
TTGTTGAGPGAGCTTA ----- GAATGATGGATGGTTGTGAATGTGTTGTGCCCCTTTCCTTTCCCCA ------- TGTTGG ---- TGG
Ca. graciliS
TTGTTGAGAGAGCATA ----- GAATGATGGATGGTTGTGAATGTGTATGTGCCCCTTTCCTTTCCCCA ------- TGTTGGTGG
Ca. spicata
TTGTTGAGAGAGCATA ----- GAATGACGGATGGTTGTGAATGTGTGTGTGCCCCTTTCCTTCCCC.r ------- TCTCGG ---- TGG
R. aurjcuia ta
TTGTTGAGAGAGCATA ----- GAATGATGGATGGTTGTGAATGTGTGTGTGCCCCTTTCCTT_CCCCA ------- TATCGGTGG
R. schneideriana
St. involucrattis TTGTTGAGAGAGTATA ----- GAATGATGGATGATTGTGAATGTGTGAGCGTGCTCCTTTCCTTGCCC ------- TGTTGG ---- TGG
CGTTGG ---- TGG
Sin. supraneanae TTGTTGAGAGAGCATA -----
TGTTGG ---- TGG
C.parviflora TGTTGG ---- TGG
C. thorel ii TTGTTGAGAGAGCATA
TTGTTGAGAGAGCATA ----- GAATGATGGATGATTGTGAATGTGTGCGTGACCCTTTCGTTAGCCCA ------- TGTTGGAACP.TGG
C. roscoeana TGTTGG ---- TGG
C. alismatifolia --- ------- TGTTGG ---- TGG
C. gracillima
TTGTTGAGAGPGCATA ----- GAATGATGGATGATTGTGAATGTGTGCGCGACCCTTTCGTTAGCCCA ------- CGTTGG ---- TGG
C. ecomata ------- TGTTGG ---- TGG
NJ TTGTTGAGAGAGCATA _TAGAATGATGGATGAATGTGAATGTGTGCGTG0CTTTCTTT
NJ C. harmaridi i
C. cf. austraiasiCa TTGTTGAGAGAGCATA ----- GATGATGGATGATTGTGAATGCGTGCGTGACCCTTTCGTTAGCCCA ------- TGTTGG ---- TGG
TTGTTGAGAGAGCATA ----- GAATGATGGATGATTGTGAATGTGTGAACGTGACCCTTTCGTCCATCGGCCCATGTTGGTGG
C.petiolata
C. aurantiaca TTGTTGAGAGAGCATAGCATAGTGATGGATGATTGTGTGTGTGJCGTGACCCTTTCGTTAGCCCATCCATGTTGGTGG
C. aeruginOSa
C. amada TTGTTGAGAGAGCATAGCATAGARTGATGGATGATTGCKAWCGTGTGCGTGACCCTTTCGTCRGCCCAKCCCATGTTGGTGG
C. amarissima TTGTTGAGAGAGCATAGCATARATGATGGATGATTGCWCGTGTGCGTGACCCTTTCGTCRGCCCATCCCRTGTTGGTGG
C. aroma tica
C. ela ta TTGTTGAGAGAGCATCATAGAATGATGGATGATTGTGICTGTGTCTTTCGTCCGCCCGTTGGTGG
C. heyneana TTGTTGAGAGAGCATAGCATAAAATGATGGATGATTGCTCGTGTGCGTGACCCTTTCRTCGGCCCATCCCATGTTGGTGG
C. longa
C. man gga TTGTTGAGAGAGCATAGCACAGTGGGATGAWTGCGCGTGTGCGTGACCCTTTTGTCGGCCCA ------- TGTTGG ----TGG
TTGTTGAGAGAGCATAGCACAGTGATGGATGATTGCGCGTGTGCGTGACCCTTTCGTCGGCCCA-------TGTTGG----TGG
C. ochrorhiza TGG
C.phaeocaulis - o
C. soloensis
C. zedoaria
C-)
C,)
140 150 160 170 180
100 110 120 130
5 67.
Ca. gracilis
Ca. spicata
R. auricula ta
R. schneideriafla
St. involucratus
Sm. supraneaflae -GGGGAGCACAA
C. pa rvi flora GCGATT
C. thorelii
GCGATT_GACCGTAGCTCGGTGCGATCCTA TTTG TAGC
C. roscoeana
NJ C. alisma tifolia
NJ C. gracillima GCGATT-GACTA--- CGGTGCGATCGGCACTAGGCT TC AGGGGCCCTTTGTGAGC -GGGGAGCCCAA
C. ecoma Ca
C. harmandii
C. cf. australasica
GCGATT_GACCGTAGCTCAGTGCGATCCT TTT GGCC
C. petiola Ca
GCGATT_GACTG_AGCTCGGTG0GATGCT TTTA
C. aurantiaCa
C. aeruginosa TTG GCCTTGTG -GGGGAGCCCI½A
C. ama da GCGATT _ GAC CGTAGCTCGGTGCGATCGG CTAAGA
C. amarissima CTAG TTG AGIGGCCCC -TTAGCGTGAGC- -GGGGAGCCCAA
GCGATT _GACCGTASCTCGSTGCGATCGG
C. a roma Ci ca
C. ela Ca
C. heyneana
C. longa -GGGGAGCCCAA
GCGAAT _GACCGTAGCTCGGKGCGAT0GGCACT
C. man gga -GGGGZGCCCAA
C. och rorh i za
C. phaeocaulis 0TTG0 CT
GCGATT_GACCGTAGCTCGGTGCGAT T
C. soloensis CTTGG TTAGCGTGAGC -GGGGAGCCCAA
C. zedoa ri a GCGATT _ GACCGTAGCTCGGTGCGATCGGCACTA GAA
cn
240 250 260 270
200 210 220 230
190
ITS2
22
. 12
8. .9 0. .
0
C,)
460 470
3 . 333
2 . 345
Ca.gracilis GTG__TCCATCA"AATTGT [415]
Ca. spicata GTG__TCCATCA - AATTGT [415]
R. auriculata GTG__TCCATCATTGT [412)
R. schneideriafla GTG_-TCCATCAITTGT [415]
St.invoiucratuS GTG__TCCATCA --- AATTGT [417]
Sm. supraneaflae GTG__TCNTCATTTGT (415)
C.parvifiora GTG_-TCAATCATCATTTGC [419)
C. thoreiii GTG--TCAATCA-TCATTTGC [420]
C. roscoeana GTG--TCAATCATTTGC [421)
C.alismatifOlia GTG--TCAT.TCATCATTCGC [419)
C. gracillima GTG_-TCPATCA-TCATTTG0 [419]
C. ecomata GTG_-TCAATCA ---- TTTGT [409]
C. harmandii GTG--TCATCATCATTTGC [419)
NJ GTG--TCAATCATTTGC [417)
C.cf.austraiaSiCa
C.petioiata GCG--TCAATCATTTGC [424]
C.aurantiaCa GTG--CCAATCATTTATTTGC [447]
C.aeruginoSa GCG--TCAATCA - "TTTGC [4251
C.arnacia GCG--TCPATCA -- TTTGC [431]
C. amarissima GCG--TCAATCATTTGC [4301
C.aromatica GCGCGTCTCATTTGC [437]
C.eiata GCGCGTCAATCATTTGC [437]
C. heyneana GCG--TCAATCATTTGC [430]
C. longa GCG--TCAPtTCA -- TTTGC [4371
C.mangga GCG--TCAATCA -- TTTGC [425]
C.ochrorhiza GCG--TCAATCATTTGC [4251
C.phaeocaulis GCG- - TCAATCA ---- TTTGC [431]
C.soioensis GCG--TCAATCA - TTTGC [429)
C. zedoaria GCG--TCAATCATTTGC [428]
cn
APPENDIX 3. Sequence data matrix (displayed from 5' to 3') of aligned ITS1 and ITS2 regions of 28 taxa of
Zingiberaceae. Numbers in bold italic (1 to 35) indicate the number and position of alignment gaps. Uncertain nucleotide
K=G/T, M=A/C, R=A/G, S=C/G, W=A/T, Y=CIT,
states are coded based on PAUP conventions (Swofford 1993) as follows:
Square brackets at the end of sequences show the real spacer length of ITS1 plus ITS2 regions.
N=AJT/GIC.
50 60 70 80 90
10 20 30 40
1.
ATCCTGAGCCAAATCCTTAGTTTGATAAAACTAAGGTTTATCAAA
TGGTAACTTCCAAATTCAGAGAAACCCTGGAATTTAAAATGGGCA TATCAP'A
Ca. spicata TGGTAACTTCCAAATTCAGAGAAACCCTGGAATTTAAAATGGGCAATCCTGAGCCAAATCCTTAGTTT ---------------
R. humeafla TCCTGAGCCAAATCCTTAGTTTGATAAACCTTAGTTTTATCAAA
TGGTAACTTCCAAATTCAGAGAAACCCTGGAATTTAAAATGGGCAA
Sm. supraflea nee TCCTGAGCCAAATCCTTAGTTTGATAAACCTTAGTTTTATCAAA
C. thorelii T GGTAACTTCCAAATTCAGAGAAACCCTGGAATTTAAAATGGGCAA CTGAGCCAAATCCTTAGTTTGATAAACCTTAGTTTTATCAAA
TC
C.rosCoeafla T GGTAACTTCCAAATTCAGAGAAACCCTGGAATTTAAAATGGGCAA
GCAATCCTGAGCCAAATCCTTAGTTTGATAAACCTTAGTTTTATCAAA
TGGTAACTTCCAAATTCAGAGAAACCCTGGAATTTAAAATGG
C.alismatifolia TCCTGAGCCAAATCCTTAGTTTGATAAACCTTAGTTTTATCAAA
TGGTAACTTCCAAATTCAGAGAAACCCTGGAATTGAAAATGGGCAA
C. gracillima TGGTAACTTCCAAATTCAGAGAAACCCTGGAATTTAAAATGGGCAATCCTGAGCCAAATCCTTAGTTT --------------- TATCAP.A
C. ecoma ta --------------------------- TATCAAA
C. harmandii TCCTGAGCCAAATCCTTAGTTTGATAAACCTTAGTTTTATCAAA
TGGTAACTTCCAAATTCAGAGAAACCCTGGAATTTAAAATGGGCAA
C. cf. australeSiCe CTGAGCCAAATCCTTAGTTTGATAAACCTTAGTTTTATCAAA
C.petiolata T GGTAAc TTCCAAATTCAGAGAAACCCTGGAATTTAAAATGGGCAATC CTGAGCCAAATCCTTAGTTTGATAAACCTTAGTTTTATCAAA
TGGTAACTTCCAAATTCAGAGAAACCCTGGAATTTAAAATGGGCAATC
C. ochrorhiza CCTGAGCCAAATCCTTAGTTTGATAAACCTTAGTTTTATCAAA
TGGTAACTTCCAAATTCAGAGAAACCCTGGAATTTAAAATGGGCAAT
C. aerugiflOSa TCCTGAGCCAAATCCTTAGTTTGATAAACCTTAGTTTTATCAAA
TGGTAACTTCCAAATTCAGAGAAACCCTGGAATTTAAAATGGGCAA
C.phaeoCauliS CTGAGCCAAATCCTTAGTTTGATAAACCTTAGTTTTATCAAA
TGGTAACTTCCAAATTCAGAGAAACCCTGGAATTTAAAATGGGCAATC
C. aurantiaCa TCCTGAGCCAAATCCTTAGTTTGATAAACCTTAGTTTTATCAAA
TGGTAACTTCCAAATTCAGAGAAACCCTGGAATTTAAAATGGGCAA
C. heyneafla TGAGCCAAATCCTTAGTTTGATAAACCTTAGTTTTATCAAA
TGGTAACTTCCAAATTCAGAGAAACCCTGGAATTTAAAATGGGCAATCC
C. longa CTGAGCCAAATCCTTAGTTTGATAAACCTTAGTTTTATCAAA
TGGTAACTTCCAAATTCAGAGAAACCCTGGAATTTAAAATGGGCAATC
C. amade TGAGCCAAATCCTTAGTTTGATAAACCTTAGTTTTATCAAA
TGGTAACTTCCAAATTCAGAGAAACCCTGGAATTTAAAATGGGCAATCC
C. zedoaria TCCTGAGCCAAATCCTTAGTTTGATAAACCTTAGTTTTATCAAA
C. zanthorrhiZa TGGTAACTTCCAAATTCAGAGAAACCCTGGAATTTAAAATGGGCAA
CTGAGCCAAATCCTTAGTTTGATAAACCTTAGTTTTATCAAA
TGGTAACTTCCAAATTCAGAGAAACCCTGGAATTTAAAATGGGCAATC
C. soloensis CTGAGCCAAATCCTTAGTTTGATAAACCTTAGTTTTATCAAA
TGGTAAATTCCAAATTCAGAGAAACCCTGGAATTTAAAATGGGCAATC
C. aroma tica CTGAGCCAAATCCTTAGTTTGATAAACCTTAGTTTTATCAAA
TGGTAACTTCCAAATTCAGAGAAACCCTGGAATTTAAAATGGGCAATC
C.purpuraSCefls TGAGCCAAATCCTTAGTTTGATAAACCTTAGTTTTATCAAA
TGGTAACTTCCAAATTCAGAGAAACCCTGGAATTTAAAATGGGCAATCC
C. elate TGGTAACTTCCAAATTCAGAGAAACCCTGGAATTTAAAATGGGCAATCCTGAGCCAAATCCTTAGTTTGATAAACCTTAGTTTTATCAAA
C. colorata
140 150 160 170 180
100 110 120 130
2. 345.
CTAGAATAAAAAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGAAGTTGACTACGTTTCGTTGGTAGTTGGAATC
Ca spi ca ta
.
R. humeana
Sm. supraneaflae
C. th orel ii
C. roscoeafla CTAGAATAAAAAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGAAGTTGACTACGTTTCGTCGGTAGTTGGAATC
C. alisma tifolia
C. gra ciii ima
C. ecoma ta
C. harmandii CTAGAATAAAAAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGAAGTTGACTACGTTTCGTCGGTAGTTGGAATC
C. cf. australasica
CTAGAATAAAAAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGAAGTTGACTACGTTTCGTCGGTAGTTGGAATC
C.petioia ta
C. ochrorhiza
C. aeruginoSa
C. phaeoca uiis CTAGAATAAAAAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGAAGTTGACTACGTTTCGTCGGTAGTTGGAATC
C. aurantiaca
C. heyneana
C. 1 onga CTAGPA-AAPAAAA
C. amada CTAGAA-PAAPPJ--- GGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGAAGTTGACTACGTTTCGTCGGTAGTTGGAATC
C. zedoa ri a
C. zan thorrhi za
C. soioensis
C. a roma tics
C. purpurascenS
CTAGAA-AAAAPAA--- GGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGAAGTTGACTACGTTTCGTCGGTAGTTGGAATC
C. el a ta
C. co 1 ora ta CTAGAA-AAAAMAA
230 240 250 260 270
190 200 210 220
CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
Ca. spi ca ta
CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
R. h urneana
CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
Sin. supraneanae
CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
C. thorel ii
CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
C. roscoeana
CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
C. alisina tifolia
CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
C. graciiiiina
CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
C. ecoma ta
CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
C. ha rmancii i CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
C. cf. australasiCa CGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
C.petiola ta CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATA
CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
C. och rorhi za
CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
C. aerugiflOsa
CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
C.phaeoCaUiiS
CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
C. a u rant .1 a ca
CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
C. heyneana
CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
C. I onga
CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
C. ama da
CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
C. zedoa na
CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
C. zan thorrhiza
CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
C. soloensiS
CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
C. a roma t I ca
CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
C. purpurascenS
CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
C. ela ta
CGTCTATCAAAATTACAGAAAAGATGTTCCTATATACCTAATACATACGTATACATACTGACATATCAAATCAAACGATTAATCATGACT
C. colorata
- o
0
C,)
330 340 350 360
290 300 310 320
280
TTATTGTGAATCCAATCCAATGGAAGTCGAAAGAAGA
CGAATCCATTATATTATATGGATAATTATAATATGAAAAATTCAGAATTAG AG
Ca. spicata AGTTATTGTGAATCCAGTCCAATGGAAGTTGAAAGAAGA
CGAATCCATTATATTATATGGATAATTATAATATTAAAAATTCAGAATTAG
R. hurneana AGTTATTGTGAATCCAGTCCGATGGAAGTTGAAAGAAGA
CGAATCCATTATATTATATGGATAATTATAATATGAAAAATTCAGAATT AG
Sm. supraneaflae TATTGTGAATCCAGTCCGATGGAAGTTGAAAGAAGA
CGAATCCATTATATTATATGGATAATTATAATATGAAAAATTCAGAATTAGAGT
C. thorelii TTATTGTGAATCCAGTCCGATGGAAGTTGAAAGAAGA
CGAATCCATTATATTATATGGATAATTATAATATGAAAAATTCAGAATTAGAG
C. roscoeafla TATTGTGAATCCAGTCCGATGGAAGTTGAAAGAAGA
CGAATCCATTATATTATATGGATAATTATAATATGAAAAATTCAGAATTAGAGT
C. alismatifolia ATTGTGAATCCAGTCCGATGGAAGTTGAAAGAAGA
CGAATCCATTATATTATATGGATAATTATAATATGAAAAATTCAGAATTAGAGTT
C. graciiliina TATTGTGAATCCAGTCCGATGGAAGTTGAAAGAAGA
CGAATCCATTATATTATATGGATAATTCTAATATGAAAAATTCAGAATTAGAGT
C. ecoma ta TTATTGTGAATCCAGTCCGATGGAAGTTGAAAGAAGA
CGAATCCATTATATTATATGGATAATTATAATATGAAAAATTMAGAATTAGAG
C. harmandii TATTGTGAATCCAGTCCGATGGAAGTTGAAAGAAGA
CGAATCCATTATATTATATGGATAATTATAATATGAAAAATTCAGAATTAGAGT
t'J C. cf. australasica TTGTGAATCCAGTCCGATGGAAGTTGAAAGAAGA
CGAATCCATTATATTATATGGATAATTATAATATGAAAAATTCAGAATTAGAGTTA
C.petiolata ATTGTGAATCCAGTCCGATGGAAGTTGAAAGAAGA
CGAATCCATTATATTATATGGATAATTATAATATGAAAAATTCAGAATTAGAGTT
C. ochrorhiZa ATTGTGAATCCAGTCCGATGGAAGTTGAAAGAAGA
CGAATCCATTATATTATATGGATAATTATAATATGAAAAATTCAGAATTAGAGTT
C. aeruginOSa AGTTATTGTGAATCCAGTCCGATGGAAGTTGAAAGAAGA
CGAATCCATTATATTATATGGATAATTATAATATGAAAAATTCAGAATTAG
C.phaeocauliS TTGTGAATCCAGTCCGATGGAAGTTGAAAGAAGA
CGAATCCATTATATTATATGGATAATTATAATATGAAAAATTCAGAATTAGAGTTA
C. aurantiaCa TGAATCCAGTCCGATGGAAGTTGAAAGAAGA
CGAATCCATTATATTATATGGATAATTATAATATGAAAAATTCAGAATTAGAGTTAT T G
C. heyneana TTGTGAATCCAGTCCGATGGAAGTTGAAAGAAGA
CGAATCCATTATATTATATGGATAATTATAATATGAAAAATTCAGAATTAGAGTTA
C. longa ATTGTGAATCCAGTCCGATGGAAGTTGAAAGAAGA
CGAATCCATTATATTATATGGATAATTATAATATGAAAAATTCAGAATTAGAGTT
C. amada TTATTGTGAATCCAGTCCGATGGAAGTTGAAAGAAGA
CGAATCCATTATATTATATGGATAATTATAATATGAAAAATTCAGAATTAGAG
C. zedoaria TTGTGAATCCAGTCCGATGGAAGTTGAAAGAAGA
CGAATCCATTATATTATATGGATAATTATAATATGAAAALATTCAGAATTAGAGTTA
C. zanthorrhiza TGAATCCAGTCCGATGGAAGTTGAAAGAAGA
CGAATCCATTATATTATATGGATAATTATAATATGAAAAATTCAGAATTAGAGTTAT T G
C. soloensiS TTGTGAATCCAGTCCGATGGAAGTTGAAAGAAGA
CGAATCCATTATATTATATGGATAATTATAATATGAAAAATTCAGAATTAGAGTTA
C. aroma tics TGAATCCAGTCCGATGGAAGTTGAAAGAAGA
CGAATCCATTATATTATATGGATAATTATAATATGAAAAATTCAGAATTAGAGTTATTG
C. purpuraSCenS TGAATCCAGTCCGATGGAAGTTGAAAGAAGA
CGAATCCATTATATTATATGGATAATTATAATATGAAAAATTCAGAATTAGAGTTAT T G
C. elata CGAATCCATTATATTATATGGATAATTATAATATGAAAAATTCAGAATTAGAGTTATTGTGAATCCAGTCCGATGGAAGTTGAAAGAAGA
C. colorata
400 410 420 430 440 450
370 380 390
TTAATCGGACGAGAATAAAGAGAG
ATTGAATATTCAATTCAATTATTAAATCATTCATTCCAGAGTTTGATAGATCTTTTG AAA AACTGA
Ca. spicata
R. humeana ATTGAATATTCAATT -----
ATTGAATATTCAATTCAATTACTAATCTTCATTCCAGAGTTTGATAGATCTTTTGC TGAT
Sm. supraneaflae TTTGAAAAACTGATTAATCGGACGAGAATAAAGAGAG
ATTGAATATTCAATTCAATTACTAAATCATTCATTCCAGAGTTTGATAGATCT
C. thoreiii AAACTGATTAATCGGACGAGAATAAAGAGAG
ATTGAATATTCAATTCAATTATTAAATCATTCATTCCAGAGTTTGATAGATCTTTTGAA
C. roscoeafla ACTGATTAATCGGACGAGAATAAAGAGAG
ATTGAATATTCAATTCAATTATTAAATCATTCATTCCAGAGTTTGATAGATCTTTTGAAAA
C. alismatifo-Lia TTTTGAAAAACTGATTAATCGGACGAGAATAAAGAGAG
C. gracillima ATTGAATATTCAATTCAATTACTAAATCATTCATTCCAGAGTTTGATAGATC
AACTGATTAATCGGACGAGAATAAAGAGAG
ATTGAATATTCAATTCAATTACTAAATCATTCATTCCAGAGTTTGATAGATCTTTTGAAA
C. ecomata
ATTGAATATTCAATTCAATTATTAAATCATTCATTCCAGAGTTTGATAGATCTTTTGAAAAACTGATTAATCGGACGAGAATAAAGAGAG
C. harmandii AAAAACTGATTAATCGGACGAGAATAAAGAGAG
ATTGAATATTCAATTCAATTATTAAATCATTCATTCCAGAGTTTGATAGATCTTTTG
C. cf. australasiCa ACTGATTAATCGGACGAGAATAAAGAGAG
ATTGAATATTCAATTCAATTATTAAATCATTCATTCCAGAGTTTGATAGATCTTTTGAA AA
C.petioia ta ACTGATTAATCGGACGAGAATAAAGAGAG
ATTGAATATTCAATTCAATTATTAAATCATTCATTCCAGAGTTTGATAGATCTTTTGAAAA
C. ochrorhiza AAAAACTGATTAATCGGACGAGAATAAAGAGAG
C. aeruginosa ATTGAATATTCAATTCAATTATTAAATCATTCATTCCAGAGTTTGATAGATCTTTTG
ATTGAATATTCAATTCAATTATTAAATCATTCATTCCAGAGTTTGATAGATCTTTTGAAAAACTGATTAATCGGACGAGAATAAAGAGAG
C.phaeocauliS
ATTGAATATTCAATTCAATTATTAAATCATTCATTCCAGAGTTTGATAGATCTTTTGAAAAACTGATTAATCGGACGAGAATAAAGAGAG
C. aurantiaCa
ATTGAATATTCAATTCAATTATTAAATCATTCATTCCAGAGTTTGATAGATCTTTTGAAAAACTGATTAATCGGACGAGAATAAAGAGAG
C. heyneana
ATTGAATATTCAATTCAATTATTAAATCATTCATTCCAGAGTTTGATAGATCTTTTGAAAAACTGATTAATCGGACGAGAATAAAGAGAG
C. longa
ATTGAATATTCAATTCAATTATTAAATCATTCATTCCAGAGTTTGATAGATCTTTTGAAAAAC TGATTAATCGGACGAGAATAAAGAGAG
C. amada
ATTGAATATTCAATTCAATTATTAAATCATTCATTCCAGAGTTTGATAGATCTTTTGAAAAACTGATTAATCGGACGAGAATAAAGAGAG
C. zedoaria
ATTGAATATTCAATTCAATTATTAAATCATTCATTCCAGAGTTTGATAGATCTTTTGAAAAAC TGATTAATCGGACGAGAATAAAGAGAG
C. zanthorrhiZa
ATTGAATATTCAATTCAATTATTAAATCATTCATTCCAGAGTTTGATAGATCTTTTGAAAAAC TGATTAATCGGACGAGAATAAAGAGAG
C. soloensis
ATTGAATATTCAATTCAATTATTAAATCATTCATTCCAGAGTTTGATAGATCTTTTGAAAAAC TGATTAATCGGACGAGAATAAAGAGAG
C. aroma tica
ATTGAATATTCAATTCAATTATTAAATCATTCATTCCAGAGTTTGATAGATCTTTTGAAAAACTGATTAATCGGACGAGAATAAAGAGAG
C.purpuraSCeflS
ATTGAATATTCAATTCAATTATTAAATCATTCATTCCAGAGTTTGATAGATCTTTTGAAAAA CTGATTAATCGGACGAGAATAAAGAGAG
C.elata ATTGAATATTCAATTCAATTATTAAATCATTCATTCCAGAGTTTGATAGATCTTTTGAAAAACTGATTAATCGGACGAGAATAAAGAGAG
C. cobra ta
490 500 510 520 530 540
460 470 480
.7
CGTCGACTTTAGAAATCGTGAGGGTTCAAGTCC
AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTTATAGTAAGAGGAAAA T C
Ca. spicata AAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCC
AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTTATAGTAAGAGGA
R. humeana
AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTTATAGTAAGAGGAAAATCCG TCGACTTTAGAAATCGTGAGGGTTCAAGTCC
Sm. supraneaflae
AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTTATAGTAAGAGGAAAA T C CGTCGACTTTAGAAATCGTGAGGGTTCAAGTCC
C. thorelii AATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCC
AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTTATAGTAAGAGGAA
C. roscoeana TCGACTTTAGAAATCGTGAGGGTTCAAGTCC
C. alismatifolia AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTTATAGTAAGAGGAAAATCCG
ATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCC
C. gracillima AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTTATAGTAAGAGGAAA
AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTTATAGTAAGAGGAAAATCCG TCGACTTTAGAAATCGTGAGGGTTCAAGTCC
C. ecomata
C. harmandii TCGACTTTAGAAATCGTGAGGGTTCAAGTCC
C. cf.austraiaSiCa AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTTATAGTAAGAGGAAAATCCG
AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTtATAGTAAGAGGAAAaTCCG T Cg ACtTtAGAAATCGTGAGGGTTCAAGTCC
C.petioiata
AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTTATAGTAAGAGGAAAATCCG TCGACTTTAGAAATCGTGAGGGTTCAAGTCC
C. ochrorhiza
-1 AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTTATAGTAAGAGGAAAATCCG T C GACTTTAGAAATCGTGAGGGTTCAAGTCC
C. aeruginoSa
AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTTATAGTAAGAGGAAAATCCG TCGACTTTAGAAATCGTGAGGGTTCAAGTCC
C. ph a eo ca u ii s
AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTTATAGTAAGAGGAAAATCCG T CG ACTTTAGAAATCGTGAGGGTTCAAGTCC
C. au rant: iaCa
AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTTATAGTAAGAGGAAAATCCG TCGACTTTAGAAATCGTGAGGGTTCAAGTCC
C. heyneana
AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTTATAGTAAGAGGAAAATCCGTCG ACTTTAGAAATCGTGAGGGTTCAAGTCC
C. longa
AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTTATAGTAAGAGGAAAATCCG T CG ACTTTAGAAATCGTGAGGGTTCAAGTCC
C. amada
AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTTATAGTAAGAGGAAAATCCG T CG ACTTTAGAAATCGTGAGGGTTCAAGTCC
C. zedoaria
AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTTATAGTAAGAGGAAAA TCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCC
C. zanthorrhiza
AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTTATAGTAAGAGGAAAATCCGTCG ACTTTAGAAATCGTGAGGGTTCAAGTCC
C. soloensis
AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTTATAGTAAGAGGAAAATCCGTCG ACTTTAGAAATCGTGAGGGTTCAAGTCC
C. aroma tica
AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTTATAGTAAGAGGAAAATCCGTCGAC TTTAGAAATCGTGAGGGTTCAAGTCC
C.purpuraSCeflS
AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTTATAGTAAGAGGAAAATCCGTCG AC TTTAGAAATCGTGAGGGTTCAAGTCC
C. ela ta
AGTCCCATTCTACATGTCAATACCGACAACAATGAAATTTATAGTAAGAGGAAAATCCGTCG AC TTTAGAAATCGTGAGGGTTCAAGTCC
C. colorata
Ii
580 590 600 610 620 630
550 560 570
111 .
012.
Ca. spica ta
CTCTTCCCCAATAAAAA_GGTAATTTTACTTTTATTTAT ------ CCTCCTTTTTCTTTT_CATCCGCGATTCAGTTC
R. hwneana
CTCTATCCCCATAAAA_GGTITTTTACTTTTATTTAT ------ CCTCCTTTTTTTTCATCAGCGATTCAGTT0
Sin. supraneanae
CTCTATCCCChATAAAAGGTTTTTACTTTTATTTAT ------ CCTCCTTTTTTTTTCATCAGCGATTCAGTTCAC
C. thorelii
CTCTATCCCCAAAAAAGGTTTTTTTTTATTTAT ------ CCTCCTTTTTTTTCATCAGCGTTCAGTTC
C. roscoeana
CTCTATCCCCAATAAAAGGTAATTTTACTTC0TTATTTAT ------ CCTCCTTTTTTTTTTCATCAGCGATTCAGTTCAC
C. a1isinatifoLia
CTCTATCCCCAATAAAGGTTTTTACTTTATATTTAT ------ CCTCCTTTTTTTTTTTCATCAGCGATTCAGTTCAC
C. gracillima
CTCTATCCCCAATAAA-A_GGTAATTTTACTTTWTATTTAT ------ CCTCCTTTTTTTTTT_CATCAGCGATTCGTTCC
C. ecornata
CTCTATCCCCAATJAAAA_GGTIATTTT1CTTTTATTTAT ------ CCTCCTTTTTTTTTT_CATCAGCGTTCAGTTcC
tQ C. harrnandii
CTCTATCCCCAATAAAAGGTTTTTTTCCTATATTTAT ------ CCTCCTTTTTTTTTT-CATCAGCGATTCAGTTCAC
C. cf. australasiCa
00 CCTCTTCCCAATAAAA_GGTAATTTTACTTTTATTTAT ------ CCTCCTTTTTTTTTT_CATCAGCGPTTCAGTTCWC
C.petioiata
CTCTATCCCCAATAAGGTTTTTACTTTTATTTAT ------ CCTCCTTTTTTTTTT-CATCAGCGATTCAGTTCC
C. ochrorhiza
CTCTATCCCCAATAAAAAAGGTAATTTTACTTCCTAAATATTTAT ------ CCTCCTTTTTTTTTT_CATCAGCGTTCAGTTCAC
C. aeruginosa
CTCTATCCCCPATAAAAAGGTTTTTACTTCCTATATTTAT ------ CCTCCTTTTTTTTTCPTCAGCGATTCAGTTCA0
C.phaeocauiis
CTCTATCCCCAATAAAAGGTAATTTTACTTCCTTATTTAT ------ CCTCCTTTTTTTTTT_CATCAGCGTTCAGTT0C
C. aurantiaca
CTCTTCCCCAATAGGTAATTTTACTTC0TATATTTAT ------ CCTCCTTTTTTTTTT-CATCAGCGATTCAGTTCAC
C. heyneana
CTCTATCCCCAATAAAAAGGTAATTTTACTTTTATTTAT ------ CCTCCTTTTTTTTTTCATCAGCGATTCAGTTCC
C. longa
CTCThTCCCCAATA/AA_GGTAATTTTACTTCCTATATTTAT ------ CCTCCTTTTTTTTT--CATCGCGATTCAGTT0AC
C. arnada
CTCTATCCCCATAAAAAGGTAATTTTACTTTTATTTAT ------- CCTCCTTTTTTTTTTCATCAGCGATTCAGTTCC
C. zedoaria
CTCTATCCCCAATAAAAA_GGTTTTTACTTTTATTTAT ------ CCTCCTTTTTTTTTT-CATCGCGTTCAGTTCAC
C. zanthorrhiza
CTCTATCCCCATAAAAAGGTAATTTTACTT0CTATATTTAT ------ CCTCCTTTTTTTTTTCATCAGCGATTCAGTTCC
C. soloensis
CTCTATCCCCAATAAAAA_GGTAATTTTACTTCCTTATTTAT ------ CCTCCTTTTTTTTTT-CATCAGCGATTCPGTTCAC
C. aroma tica
CTCTATCCCCAATAAAGGTAATTTTACTTCCTATATTTAT ------ CCTCCTTTTTTTTTTCATCAGCGATTCAGTTCC
C. purpurascens
CTCTATCCCCAATAAAGGTAATTTTACTTCCTTATTTAT ------ CCTCCTTTTTTTTTT-CATCAGCGATTCAGTTCAC
C. ela ta
CTCTATCCCCAATAAAAGGTAATTTTACTTCCTTATTTAT ------ CCTCCTTTTTTTTTT-CATCAGCGATTCAGTTCC
C. colorata
tj
670 680 690 700 710 720
640 650 660
AATTCACTATCTTTCTCATTCACTCCACTCTTTCACAACACAAATGTATCCGAACTAAAATCCTTGGAT C TTATCCCAATTTCGATAGAT
Ca. spicata
EL humeana AATTCACTATCTTTCTCATTCACTCCACTCTTTCACAACACAAATGTATCCGAACTAAAATCCCTGGA T C TTATCCCAATTTCGATAGAT
Sm. supraneaflae AATTCACTATCTTTCTCATTCACTCCACTCTTTCACAACACAAATGTATCCGAACTAAAATCCTTGG AT C TTATCCCAATTTCGATAGAT
C. thoreiii AATTCACTATCTTTCTCATTCACTCCACTCTTTCACAACACAAATGTATCCGAACTAAAATCCTTGGATCTTATCCCAATTTCGATAGAT
C. roscoeafla AATTCACTATCTTTCTCATTCACTCCACTCTTTCACAACACAAATGTATCCGAACTAAAATCCTTGGATCTTATCCCAATTTCGATAGAT
C. alismatifolia AATTCACTATCTTTCTCATTCACTCCACTCTTTCACAACACAAATGTATCCGAACTAAAATCCTTGGA T C TTATCCCAATTTCGATAGAT
C. gracillima AATTCACTATCTTTCTCATTCACTCCACTCTTTCACAACACAAATGTATCCGAACTAAAATCCTTGGATCTTATCCCAATTTCGATAGAT
C. ecomata AATTCACTATCTTTCTCATTCACTCCACTCTTTCACAACACAAATGTATCCGAACTAAAATCCTTGGATCTTATCCCAATTTCGATAGAT
C. harmandii AATTCACTATCTTTCTCATTCACTCCACTCTTTCACAACACAAATGTATCCGAACTAAAATCCTTGGATCTTATCCCAATTTCGATAGAT
C. cf. australasica AATTCACTATCTTTCTCATTCACTCCACTCTTTCACAACACAAATGTATCCGAACTAAAATCCTTGGATCT TATCCCAATTTCGATAGAT
t'J
C.petioiata AATTCACTATCTTTCTCATTCACTCCACTCTTTCACAACACAAATGTATCCGAACTAAAATCCTTGGATCTTATCCCAATTTCGATAGAT
L..)
C. ochrorhiza AATTCACTATCTTTCTCATTCACTCCACTCTTTCACAACACAAATGTATCCGAACTAAAATCCTTGGATCT TATCCCAATTTCGATAGAT
C. aeruginosa AATTCACTATCTTTCTCATTCACTCCACTCTTTCACAACACAAATGTATCCGAACTAAAATCCTTGGATCT TATCCCAATTTCGATAGAT
C. pha eoca ul i s AATTCACTATCTTTCTCATTCACTCCACTCTTTCACAACACAAATGTATCCGAACTAAAATCCTTGGATCT TATCCCAATTTCGATAGAT
C. aurantiaca AATTCACTATCTTTCTCATTCACTCCACTCTTTCACAACACAAATGTATCCGAACTAAAATCCTTGGA T C TTATCCCAATTTCGATAGAT
C. heyneana AATTCACTATCTTTCTCATTCACTCCACTCTTTCACAACACAAATGTATCCGAACTAAAATCCTTGGATCTTATCCC AATTTCGATAGAT
C. longa AATTCACTATCTTTCTCATTCACTCCACTCTTTCACAACACAAATGTATCCGAACTAAAATCCTTGGATCTTATCCC AATTTCGATAGAT
AATTCACTATCTTTCTCATTCACTCCACTCTTTCACAACACAAATGTATCCGAACTAAAATCCTTGGATCTTATCCC AATTTCGATAGAT
C. amada
C. zedoaria AATTCACTATCTTTCTCATTCACTCCACTCTTTCACAACACAAATGTATCCGAACTAAAATCCTTGGATCTTA T C CCAATTTCGATAGAT
C. zanthorrhiza AATTCACTATCTTTCTCATTCACTCCACTCTTTCACAACACAAATGTATCCGAACTAAAATCCTTGGATCTTATCCC AATTTCGATAGAT
C. soloensis AATTCACTATCTTTCTCATTCACTCCACTCTTTCACCAcTGTATCCGCTTCCTTGGATCTTATCCCTTTCGATAGAT
C. aroma tica AATTCACTATCTTTCTCATTCACTCCACTCTTTCACAACACAAATGTATCCGAACTAAAATCCTTGGATCTTATCCC AATTTCGATAGAT
C.purpurascenS AATTCACTATCTTTCTCATTCACTCCACTCTTTCACAACACTGTATCCGCTAAAATCCTTGGATCTTATCCCTTTCGATAGAT
C. elata AATTCACTATCTTTCTCATTCACTCCACTCTTTCACCAcTGTATCCGCTATCCTTGGATCTTATCCCTTTCGATAGAT
C. colorata AATTCACTATCTTTCTCATTCACTCCACTCTTTCACCACATGTATCCGCTTCCTTGGATCTTATCCCTTTCGATAGAT
760 770 780 790 800 810
730 740 750
ACAATACCTCTACAAATAAACATATATGGGCAAATAATCTCTATTATTGAATCATTCACAG TCCATATCATTATCCTTACGCTTACTAGT
Ca. spicata
R. humeana ACAATACCTCTACAAATAAACATATATGGGCAAATAATCTCTATTATTGAATCATTCAC AGTCCATATCATTATCCTTACGCTTACTAGT
ACAATACCTCTACAAATAAACATATATGGGCAAATAATCTCTATTATTGAATCATCCACAG TCCGTATCATTATCCTTACGCTTACTAGT
Sm. supraneaflae
C. thoreiii ACAATACCTCTACAAATAAACATATATGGGCAAATAATCTCTATTATTGAATCATTCAC AGTCCGTATCATTATCCTTACGCTTACTAGT
C. roscOeafla ACAATACCTCTACAAATAAACATATATGGGCAAATAATCTCTATTATTGAATCATTCACAG T CC GTATCATTATCCTTACGCTTACTAGT
ACAATACCTCTACAAATAAACATATATGGGCAAATAATCTCCATTATTGAATCATTCACAG TCCGTATCATTATCCTTACGCTTACTAGT
C.alismatifoiia
A CAATACCTCTACATAACATATATGGGCAATAATCTCTATTATTGTCATTCACAGTCCGTATCATTATCCTTACG CTTACG
C. gracillima
ACAATACCTCTACAAATAAACATATATGGGCAAATAATCTCTATTATTGAATCATTCACAG T CC GTATCATTATCCTTACGCTTACTAGT
C. ecomata
C. harmandii ACAATACCTCTACAAATAAACATATATGGGCAAATAATCTCCATTATTGAATCATTCACAGTCCG TATCATTATCCTTACGCTTACTAGT
A CAATACCTCTACAATACATATATGGGCATTCTCTATTATTGTCATTCACTCCGTATCATTATCCTTACGC TTACTAGT
C. cf. australasica
A CAATACCTCTACAATAACATATATGGGCATTCTCTATTATTGTCATTCACAGTCCGTATCATTATCCTTACGC TTACTAG
C.petiolata
ACAATACCTCTACAAATAACATATATGGGCATTCTCTATTATTGTCATTCACAGTCCGTATCATTATCCTT AC GCTTACG
N) C. ochrorhiza
ACAAT ACCTCTACAAATWCATATATGGGCAAATAATCTCTATTATTGTCATTCACAGTCCGTATCATTATCCTTACGCTTAC TAG T
C. aeruginosa
A CAATACCTCTACAATAAACATATATGGGCAATAATCTCTATTATTGATCATTCACAGTCCGTATCATTATCCTTACGC TTACTAGT
C.phaeocaulis
A CAATACCTCTACAAATAAACATATATGGGCAAATAATCTCTATTATTGTCATTCACAGTCCGTATCATTATCCTTACGCTTAC TAGT
C. aurantiaCa
A CAATACCTCTACAAATAAACATATATGGGCAAATAATCTCTATTATTGTCATTCACAGTCCGTATCATTATCCTTACGCTTACTAGT
C. heyneana
A CAATACCTCTACAAATAAACATATATGGGCAAATAATCTCTATTATTGATCATTCACAGTCCGTATCATTATCCTTACGCTTACTAG T
C. longa
ACAATACCTCTACAATAAACATATATGGGCAAATTCTCTATTATTGTCATTCACAGTCCGTATCATTATCCTTACGCTTACTAGT
C. amada
A CAATACCTCTACAAATAAACATATATGGGCAAATAATCTCTATTATTGTCATTCACAGTCCGTATCATTATCCTTACGCTTACTAGT
C. zedoaria
A CAATACCTCTACAAATAAACATATATGGGCAATAATCTCTATTATTGAATCATTCACAGTCCGTATCATTATCCTTACGCTTACTAG T
C. zanthorrhiza
A CAATACCTCTACAAATAAACATATATGGGCAAATAATCTCTATTATTGTCATTCACAGTCCGTATCATTATCCTTACGCTTACTAG T
C. soloensis
A CAATACCTCTACAATAAACATATATGGGCAAATAATCTCTATTATTGAATCATTCACAGTCCGTATCATTATCCTTACGCTTACTAG T
C. aroma tica
A CAATACCTCTACAATAAACATATATGGGCATATCTCTATTATTGTCATTCACAGTCCGTATCATTATCCTTACGCTTACTAGT
C. purpurascens
ACAATACCTCTACAAATAAACATATATGGGCAAATAATCTCTATTATTGAATCATTCACAGTCCG TATCATTATCCTTACGCTTACTAGT
C. ela ta
ACAATACCTCTACAAATAACATATATGGGCAAATTCTCTATTATTGTCATTCACAGTCCGTATCATTATCCTTACGCTTAC TAGT
C. colorata
rn
850 860 870 880 890 900
820 830 840
TAAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACAAACACGACACCGGGATGATGCATGGGAAATGGTCGGGATAGCTCA
Ca. spicata TAAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACAAACACTACACCAGGATGATGCATGGGAAATGGTCGGGATAGCTCA
R. humeana TAAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACAAACACTACACCAGGATGATGCATGGGAAATGGTCGGGATAGCTCA
Sm. supraneaflae TAAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACAAACACTACACCAGGATGATGCATGGGAAATGGTCGGGATAGCTCA
C. thorelii TAAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACAAACACTACACCAGGATGATGCATGGGAAATGGTCGGGATAGCTCA
C. roscoeafla TAAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACAAACACTACACCAGGATGATGCATGGGAAATGGTCGGGATAGCTCA
C. alisma tifolia TAAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACAAACACTACACCAGGATGATGCATGGGAAATGGTCGGGATAGCTCA
C. graciiliina
TAAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACAAACACTACACCAGGATGATGCATGGGAAATGGTCAGGATAGCTCA
C. ecoma ta TAAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACAAACACTACACCAGGATGATGCATGGGAAATGGTCGGGATAGCTCA
C. harmandii
TAAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACAAACACTACACCAGGATGATGCATGGGAAATGGTCGGGATAGCTCA
C. cf. australasiCa
TAAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACAAACACTACACCAGGATGATGCATGGGAAATGGTCGGGATAGCTCA
C. petioia ta TAAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACAAACACTACACCAGGATGATGCATGGGAAATGGTCGGGATAGCTCA
C. ochrorhiza
TAAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACAAACACTACACCAGGATGATGCATGGGAAATGGTCGGGATAGCTCA
C. aeruginoSa
TAAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACAAACACTACACCAGGATGATGCATGGGAAATGGTCGGGATAGCTCA
C. phaeocauiis
TAAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACAAACACTACACCAGGATGATGCATGGGAAATGGTCGGGATAGCTCA
C. aurantiaca
TAAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACAAACACTACACCAGGATGATGCATGGGAAATGGTCGGGATAGCTCA
C. heyneana
TAAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACAAACACTACACCAGGATGATGCATGGGAAATGGTCGGGATAGCTCA
C. longa
TAAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACAAACACTACACCAGGATGATGCATGGGAAATGGTCGGGATAGCTCA
C. amada TAAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACAAACACTACACCAGGATGATGCATGGGAAATGGTCGGGATAGCTCA
C. zedoaria TAAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACAAACACTACACCAGGATGATGCATGGGAAATGGTCGGGATAGCTCA
C. zanthorrhiza
TAAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACAAACACTACACCAGGATGATGCATGGGAAATGGTCGGGATAGCTCA
C. soloensiS
C. aroma tica TAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACACACTACACCAGGATGATGCATGGGTGGTCGGGATAGCC
TAAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACAAACACTACACCAGGATGATGCATGGGAAATGGTCGGGATAGCTCA
C. purpurascenS
TAAATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACAAACACTACACCAGGATGATGCATGGGAAATGGTCGGGATAGCTCA
C. elata
C. coiorata TA ATTTTTTACTACTTTTTAGTCCCTTTAATTGACATAGACACCACTACACCAGGATGATGCATGGGTGGTCGGGATAGC C
Ca.spiCata GTTGGTAGAGC [908]
R.hwneana GTTGGTAGAGC [8811
Sin.supraneaflae GTTGGTAGPGC [900]
C.thorelii GTTGGTAGAGC [901]
C.roscQeafla GTTGGTAGAGC [9001
C.alisrnatifOlia GTTGGTAGAGC [903]
C.gracilliina GTTGGTAGAGC [9031
C.ecornata GTTGGTAGAGC [887]
C.harmandii GTTGGTAGAGC [886]
C. cf.avstralasiC& GTTGGTAGAGC [903]
C.petiolata GTTGGTAGPGC [9031
C.ochrorhiza GTTGGTAGAGC [899]
C.aerugiflOSa GTTGGTAGAGC [902]
C.phaeocauliS GTTGGThGAGC [9001
C.aurantiaCa GTTGGTAGAGC [903]
C.heyrieana GTTGGTAGAGC [901]
C.longa GTTGGTAGAGC
C.amada GTTGGTAGAGC [9001
C.zedoaria GTTGGTAGAGC [8991
C.zanthorrhiza GTTGGTAGAGC [9011
C.soloensis GTTGGTAGAGC [9001
C.aromatiCa GTTGGTAGAGC
C.purpurasCefls GTTGGTAGAGC [900]
C.elata GTTGGTAGAGC [8991
C.colorata GTTGGTAGAGC [900]
- o
C)
Ill
C,,
APPENDICES
243
APPENDICES
244
APPENDICES
245
APPENDICES
246
APPENDICES
Notes:
LE1= Length of adaxial epidermis; WE1= Width of adaxial epidermis; LE2= Length of
abaxial epidermis; WE2= Width of abaxial epidermis; HS1= Height of stomatal on
adaxial; WSI= Width of stomatal on adaxial; HS2= Height of stomatal on abaxial;
WS2= Width of stomatal on abaxial.
zed: C. zedoaria; pha: C. phaeocaulis; Zan: C. zanthorrhiza 1; aer: C. aeruginosa; hey:
C. heyneana; man: C. mangga; Ion: C. longa; pur: C. purpurascens; col: C. colorata;
par: C.
Zana: C. zanthorrhiza 2; tho: C. thorelii; ros: C. roscoeana; au: C. alismatifolia;
parviflora; aur: C. aurantiaca; amar: C. amarissima; amad: C. amada.
247
APPENDIX 5 Measurements of flower in the morphometric analysis.
4 5 6 7 8 9 10 11
SPECIES 1 2 3
25.750 13.750 12.250 11.838 12.125 9.500 10.125
C.aeruginosa 3.000 12.375 24.313 7.261
18.375 14.875 9.375 16.250 17.375 5.875 13.750
C.alismatifolia 2.625 9.875 19.500 6.673
21.063 14.500 10.250 10.875 12.000 7.875 10.500
C.amada 3.500 9.750 26.250 6.280
28.500 15.250 11.375 13.625 14.375 11.063 10.750
C.amarissima 4.000 11.625 27.625 9.813
13.688 5.875 7.875 8.438 9.375 4.688 7.875
C.aromatica 2.250 6.313 16.750 4.514
20.188 14.750 8.000 17.188 19.375 8.125 11.375
C.aurantiaca 2.625 12.500 22.000 6.084
12.125 10.375 7.500 8.438 9.375 5.813 6.813
C.cfaustralasica 2.000 6.125 14.375 4.710
28.625 12.875 13.000 12.625 14.250 10.625 11.000
C.colorata 2.625 9.875 32.875 8.243
4.318 12.000 12.500 7.875 18.750 18.750 4.625 8.125
C.ecomata 2.125 13.000 21.500
24.750 12.000 11.375 12.250 14.500 9.375 11.500
C.euchroma 3.000 10.438 34.500 7.261
2.944 7.313 4.500 3.188 3.750 3.875 2.438 5.563
C.gracillima 1.250 5.125 15.500
3.925 8.750 8.875 5.250 8.750 9.625 4.625 12.625
C.harmandii 1.875 11.250 23.250
9.028 28.250 12.500 11.250 12.000 12.500 10.125 9.000
C.heyneana 2.875 17.125 32.750
6.084 22.500 10.375 10.125 9.125 10.625 6.625 8.500
C.longa 1 2.625 10.750 34.500
8.635 24.625 12.875 10.000 11.375 13.750 8.500 9.875
C.longa2 3.063 12.250 26.000
6.673 30.313 11.875 12.750 11.688 12.750 10.500 8.125
C.longa 3 2.750 10.375 31.250
7.261 29.625 12.750 12.188 12.250 12.750 9.750 8.500
C.longa 4 3.750 11.188 30.500
7.850 29.375 13.125 11.938 11.125 11.500 9.063 9.125
C.longa5 3.000 10.625 26.938
27.000 7.850 27.313 15.000 11.000 13.500 13.813 9.750 13.375
C.mangga 2.750 11.125
4.121 7.750 5.313 3.875 4.863 5.125 3.438 5.625
C.parvflora 1.813 6.125 11.688
5.888 20.125 9.125 8.375 9.000 9.125 2.750 8.625
C.petiolata 2.250 11.188 23.875
18.125 5.495 16.125 9.125 8.250 9.875 10.000 6.250 6.750
C.purpurascens 2.500 8.500
5.495 12.313 12.250 6.625 11.625 11.875 7.188 13.250
C.roscoeana 2.625 22.000 36.500
16.875 7.065 24.250 10.250 10.125 12.500 14.688 6.438 10.500
C.soloensis 3.125 8.250
15.000 1.963 5.563 6.688 4.563 6.188 6.875 3.750 10.000
C.thorelii 1.875 5.938
10.205 36.375 15.000 16.438 15.000 16.000 12.500 10.375
C.zanthorrhiza 5.000 8.250 39.625
C-)
M
co
APPENDIX 5 (continued) Measurements of flower in the morphometric analysis.
15 16 17 18 19 20 21 22
SPECIES 12 13 14
15.500 10.250 16.500 40.375 4.500 3.000 2.125 2.625
C.aeruginosa 13.063 5.750 7.688
22.500 5.875 12.375 38.375 6.250 2.313 1.438 1.875
C.alismaijfolia 16.250 3.750 3.125
17.000 7.500 13.125 43.250 4.688 2.500 1.750 2.375
C.amada 13.500 5.125 7.375
15.200 11.375 19.500 46.750 4.125 3.000 2.625 2.750
C.amarissima 15.125 6.375 9.000
10.625 5.438 8.875 28.625 2.500 1.938 1.500 1.750
C.aromalica 9.625 3.063 5.125
16.500 6.125 17.000 38.750 6.000 2.250 1.000 2.125
C.auran(iaca 14.125 5.625 7.500
8.750 3.875 10.000 24.438 3.063 1.875 0.875 1.375
C.cfausiralasica 8.063 2.625 4.250
18.500 10.375 16.500 50.500 5.000 2.500 1.750 2.500
C.colorata 15.000 7.125 9.125
15.000 5.000 10.750 32.000 8.750 2.125 1.000 1.875
C.ecomata 8.375 2.250 6.875
18.000 10.500 19.000 52.200 4.875 3.188 2.500 2.500
C.euchroma 14.700 5.500 8.438
5.250 2.250 3.875 22.188 1.563 1.188 0.750 1.063
C.gracillima 5.438 1.813 1.938
1.188 16.500 4.125 8.750 38.625 3.875 1.875 1.000 1.500
C.harmandii 13.500 0.938
7.125 16.500 11.625 15.500 45.250 4.375 2.500 2.125 2.625
C.heyneana 9.625 6.125
tQ
5.750 13.375 8.875 12.875 47.875 4.563 2.500 1.625 2.875
C.longa 1 10.750 5.000
15.500 9.688 16.500 43.063 4.188 2.625 1.625 2.625
C.longa2 14.000 5.500 8.750
6.875 14.500 12.625 17.500 44.750 4.250 2.875 2.125 2.750
C.longa3 10.750 6.500
8.125 14.500 12.625 18.000 45.500 4.438 2.500 2.125 2.875
Clonga 4 11.250 6.375
9.063 18.000 13.313 20.000 41.938 4.250 2.500 2.125 3.063
C.longa5 12.000 5.875
9.750 16.500 10.688 16.500 43.500 4.375 2.625 2.250 2.500
C.mangga 13.750 5.938
2.000 7.625 2.563 4.188 20.500 1.500 1.375 0.750 1.250
C.parviflora 7.000 1.688
6.500 10.875 8.000 12.000 36.625 3.250 1.750 1.000 1.875
C.petiolata 10.500 4.500
4.875 10.000 6.500 9.750 29.000 3.500 2.250 1.625 2.000
C.purpurascens 8.375 3.750
10.875 18.000 4.500 19.500 54.250 3.938 2.125 1.125 2.875
C.roscoeana 15.125 2.125
7.125 16.500 9.750 14.000 32.750 4.625 2.875 1.875 2.625
C.soloensis 12.750 5.188
1.188 3.563 12.000 1.875 7.500 27.500 2.188 1.188 0.563 1.063
C.thorelii 10.625
8.250 9.875 17.500 14.063 17.000 58.750 4.375 3.375 2.750 2.875
Czanthorrhiza 14.125
rri
0
C)
'ii
(I)
APPENDIX 5 (continued) Measurements of flower in the morphometric analysis.
23 24 25 26 27 28
SPECIES
0.063 2.625 4.000 2.250 31.438 0.667
C.aeruginosa
1.125 3.938 5.000 2.500 29.688 0.348
Calismatifolia
0.250 3.750 3.313 1.250 34.688 0.909
Camada
0.063 3.000 4.375 2.000 34.750 0.879
C.amarissima
0.188 2.313 2.125 1.000 21.563 0.606
C.aromatica
0.688 1.875 2.813 1.250 29.875 0.758
C.aurantiaca
0.375 1.125 3.000 0.938 18.563 0.606
C.cfaustralasica
0.188 3.500 4.000 1.688 41.375 1.212
C.colorata
1.125 6.000 2.500 1.000 36.250 0.545
C.ecomata
0.125 3.750 3.250 1.750 43.125 0.788
C.euchroma
1.188 2.313 1.438 0.938 19.375 1.273
C.gracillima
2.000 1.625 2.750 1.500 28.750 0.606
C.harmandii
0.125 2.500 4.375 1.875 39.625 0.636
C.heyneana
0.313 3.625 3.625 1.875 42.688 0.364
C.longa 1
0.500 3.750 3.938 1.688 33.938 0.818
C.longa2
0.250 2.625 4.688 2.063 38.125 1.061
C.longa 3
0.250 3.875 4.250 1.938 38.813 0.818
C.longa 4
0.375 3.375 4.313 2.063 34.563 0.909
C.longa5
0.250 4.375 4.750 2.125 35.750 0.667
C.mangga
1.688 1.125 1.813 1.188 14.313 1.970
C.parvzflora
0.313 3.000 3.125 1.250 30.125 0.636
C.petiolata
0.125 2.063 2.125 1.063 23.688 0.606
C.purpurascens
3.813 2.500 3.563 1.813 42.938 0.365
C.roscoeana
0.375 3.125 4.125 2.000 24.625 0.758
C.soloensis
1.000 1.438 1.313 0.813 18.625 0.303
C.thorelii
0.188 3.250 5.813 3.188 47.250 1.061
C.zanthorrhiza
Note: Number 1-28 reters to tne cnaracers iisLeu 111 1dIJI L
APPENDIX 6 Sequence data matrix (displayed from 5' to 3') of aligned ITS2 region of 32 accessions
representing 27 taxa of Zingiberaceae.
30 40 50 60 70 80 90
10 20
Taxon ITS2
I
3
Ca. gracilis
Ca. spica ta GAAATTGGCCTCGTGTGTCCTC--GGGCACAGTCGGTTGAAGA
R. auriculata AT --- CGTCGCTTTTGCTCCATGCATTGCTGGTGT0G
R. schneideriana
St. involucra tus
Sm. supraneanae
C.parviflora
C. thorelii
C. roscoeana
C. alisma tifolia
C. gracillima
C. ecoma ta
tQ
C. harmandii
C. cf.
C.petiola ta
C. ochrorrhiza
C. aeruginosa a
C. aeruginosa b
C.phaeocauliS a
C.phaeoCa ulis b
C. amarissima a
C. amarissima b
C. a uran tiaca ATTGCCGCCGCTTTTGCTCCATGCTTTATTAGCATTGAGC_AGCGCGTTGGCCCCGTGTGCCCTCGGGCACAGTCGGTCGGA
C. heyneana a
C. heyneana b
C. iongala
C. ion gaib
AT --- YGTCGCTTTTGCTCCATGCTYYGTYGGCATTGAG0G ---- RTTG0GTGTG0T0TC TCTC
C. ion ga2a
C. ionga2b
C. amada a
C. amada b
C,)
50 60 70 80 90
10 20 30 40
Taxon ITS2 . .3
. 2 .
1 . . . -- _CGGAAGTTGGCCCCGTGTG00CT _GGGCACAGTCGGTCGAAGA
C.zedoarial a AT-
AT-CGTCGCTTTTGCTCCATGCTTYGTCGGCATTGAGCG -- _CGGAAGTTGGCCCCGTGTG000TC _GGGCACAGTCGGTCGAAGA
C.zedoarial b AT- __CGTCGCTTTTGCTCCATGCTTCGTC GCATTGAGCG- -- _CGGAAGTTGG000CGTGTGCCCTC _GGGCACAGTCGGTCGAAGA
C. zedoaria2a AT- __CGTCGCTTTTGCTCCATGCTTCGT0 CATTAGCG- - CGGAAGTTGGCCCCGTGTGCCCTC--GGGCACAGTCGGTCGAAGA
C. zedoaria2b AT- __CGTCGCTTTTGCTCCATGCTTCGTCG ATTGAGCG - - _CGGAAGTTGGCCCCGTGTGCCCTC _GGGCACAGTCGGTCGAAGA
C. zedoaria3a AT- __CGTCGCTTTTGCTCCATGCTTCGTC CATTGAGCG- - __CGGAAGTTGGCCCCGTGTG000TC _GGGCACAGTCGGTCGAAGA
C. zedoaria3b
C.zedoaria cif. a AT- __CGTCGCTTTTRCTCCATG0TTYGTCGGTT GCG - - - _CGGAAGTTGGCCCCGTGTRCCCTC _GGGCACAGTCGRTCGAAGA
NJ
Ui C.zedoaria cf. b AT- __CGTCGCTTTTGCTCCATGCTTCGTG0ATT GCG--- - _CGGAAGTTGGCCCCGTGTRCCCTC _GGGCACAGTCGGTCGAAGA
NJ AT-- YGTSGCTTTWGCTCCATGCTTYGTCGGCATTGAGCG _CGGAAGTTGGCCCCGTGTG000TC _GGGCACAGTCGGTCGAAGA
C. zanthorrhizala
C. zanthorrhizalb AT-- _YGTSGCTTTWGCTCCATGCTTYGTC CATT GCG-- - _CGGAAGTTGGCCCCGTGTGCCCTC _GGGCACAGTCGGTCGAAGA
C. zanthorrhiza2a AT- __CGTCGCTTTTRCTCCATGCTTYGT0G ATTGAGCG- --
AT- __CGTCGCTTTTRCTCCATGCTTYGT RCATT GCG- -- _CGGAAGTTGGCCCCGTGTRCC0TC -GGGCACAGTCGRTCGAAGA
C. zanthorrhiza2b
C.soloensis a AT- __CGTCGCTTTTGCTCCATGCTTYGTCG ATTGA G- -
- - CGGAAGTTGGCCCCGTGTGCCCTC--GGGCACAGTCGGTCGAAGA
C.soloensiS b AT- __CGTCGCTTTTGCTCCATGCTTYGTGTT
TGTCGCTTTTGCTCCATGCTTTGTCG JATTGAGTG GTTGGTGTGCCCTC -GGGCACAGTCGGTCGAAGA
C.arornatiCa a GT ---
GT --- TGTCGCTTTTGCTCCATGCTTTGTCCATTGTGAG0G0TT00GTGTGCC _GGGCACAGTCGGTCGAAGA
C.aromatiCa b
130 140 150 160 170 180
100 110 120
4: 67
Ca. gracilis
Ca. spicata
R. auricula ta AGAACGT-----CCCCGTCGC -- TTT - AGGATT
GTGGG_TAGTCCGCAGTCGTCGGGC GAT TGTTGGTC CGTC
R. schneideriafla
St. jnvolucra tus
Sm. suprafleaflae
C.parviflOra
C. thorelii
C. roscoeana
C. alisma tifolia
C. gracillirna
C. ecoma ta
GTGGG_TACTCGGCAATCGTCGAG0A0GATG0GTTTT0GAG0T0GT0GTCCTCGTC
C. harrnandii
C. cf.
C.petiola ta
C. ochrorrhiza
C. aeruginosa a
C. aeruginosa b
C.phaeocauliS a
C.phaeocaulis b
C. amarissima a 0GTTTT0G0CTGTTCCTCGTCGT
GTGGG_TAGTCGRTAATCGTCGAGC
C. amarissLma b
GTGGGGTAGCCGGTAGTCGTCGAGCACGATGGATGTTGGTCGTCACGAGCGAGAACTGAACATCGT--CCTTGTCGTCGTTTCGGAACGA
C. aurantiaca
C. heyneana a
C. heyneana b
C. longal a
C. longal b
C. longa2 a
C. ion ga2 b
C. amada a m
C. amada b
C)
C,,
120 130 140 150 160 170 180
100 110
4. 567
-TTT-GGGATGA
C.zedoarial a --TTCGGGATGA
C.zedoarial b
GTGGG-TAGTCGGTAATCGTCGAGCACGATGGACGTTGGTCGTCGCGAGCGAGAACTGAACGTCGTG T CC TCGTCGT--TTT-GGGATGA
C. zedoaria2a -CCTCGTCGT- -TTT-GGGATGA
C. zedoari.a2b
C. zedoaria3a GTGGG_TAGTCGGTAATCGTCGAGCACGATGGA0GTTT0GT0G0TGcGTcGTGTccTCGTT -TTT-GGGATGA
GTGGG_TAGTCGGTAATCGTCGAGCACGATGGACGTTGGT0GT0G TGTT -CCTCGTCGT - -TTT-GGGATGA
C. zedoaria3b
-CCTCGTCGT--- TTCGGGATGA
C.zedoaria cf. a
C.zedoaria cf. b GTGGG_TAGTCGGTAATCGTCGAGCACGATGGACGTTGGT0GT0G0G0G0TG0GTcGTGTCCTCGTT -TTT-GGGATGA
-TTT-GGGATGA
C. zanthorrhizala
GTGGG_TAGTCGGYAATCGTCGAGCACGATGGACGTTGGTCGTCG0 CTGTCGT -CCTCGTCGT- -TTT-GGGATGA
C. zanthorrhizalb
GYGGG_TAGTCGGYAATCGTCGAGCACGATGGACGTTGGTCGT0 TTCGTGTTCGTT -TTT-GGGATGA
C. zanthorrhiza2a
-CCTCGTCGT--TTT-GGGATGA
C. zanthorrhiza2b
-TTT-GGGATGA
C.soloensis a
C.soloensis b
-TTT-GGGATGA
C.aromatica a
-CCTCGTYGT- -TTT-GGGATGA
C.aromatica b
rn
rn
Cn
200 210 220 230 240 250
190
. 9. . . .0 1 .2
8
GTCCTCAA ---- GAGACCCTGTGTGAT ------- TGTGATGTCGTGTGA AGTGCCGTG TCCATCA -AATTGT [2251
Ca. g-racilis GTCCTCAA ---- GAGACCCTGTGTGAT ------- TGTGATGTCGTGTGAAGTGCCGTG_TCCATCA --- TTGT [225]
Ca. spi ca ta
R.auriculata TTCCTCAA ---- GAGACCCCGTGTGAT ------- TGTGATGCGGTGTGAAGCCCCCTG_TCCATCA_.TTGT [222]
GTCCTCAA__-- GAGACCCCGTGTGAT ------- TGTGATGTCGTGCGAAGTGCCCTGTCCATCATTGT [2211
R. schneideriafla
GACCTCAA ---- GAGACCCTGTGTGAT ------ TTGCGGAGTCGGGTGAAGTGCCGTG_TCCATCATTGT [227]
St. involuCra tus
GTCCTCAA ---- GAGACCCTGTGTGAT ------ TTGCGGAGTCGGACGPAAGTGCCGTG_TCTCA ---- TTTGT [226]
Sm. supraneanae
GTCCTCAA ---- GAGACCCTGTGTGAT ------- TGTGATGTCGTGTGAAAGTGCCGTGTCCATCATTGT [226]
C.parvifiora
GTCCTCAA ---- GAGA000TATGTGAT ------- TGCAGAGTCGGACGAAGCGCTGTGTCTCATCATTTGC [228]
C. thorelii [228]
C. rosCOeafla
C. al isma tifolia GTCCTCAA ---- GAGACCCTACGTGAT ------- TGCAGAGTCGGATGAAGCGCTGTGTCTCATCATTCGC [228]
GTCCTCAA ---- GAGACCCTATGTGAT ----- -_TGCAGAGTCGGACGAAGCGCTGTGT0T0TTTTGC [228]
C. graCillima
C. ecomata GTCCTCAA__ -- GAGACCCTGTGTGAT ------- TGCGGAGTCGGTTGAAGTGCCGTGTCTCATTTGT [225]
C. harmanciii GTCCTCAA ---- GAGACCCTATGTGAT ------- TGCAGAGTCGGATGAAGCGCTGTGTCATCATCATTTGC [227]
[229]
C. [231]
C.petiola ta
GTCCTCCA ---- GAGACCCTGTGTGAT ---- TTGCGGAGTCGTGGCGCCGCGTCTCJTTTGC [230]
C. ochrorrhiza TTTGC [228]
GTCCTCAA ---- GAGACCCTGTGTGAT ---- ATT AGTCGCGTG GCGCCGCG TCAATCA
tQ C. aeruginosa a
GTCCTCCA ---- GAGACCCTGTGTGAT RATWGCGSAGTCGTWAGCGCCGCGTC1TC]TTTGC [230]
Li C.aeruginoSa b TTTGC [228]
GCCCTCA ---- GAGACCCTGTGTGAT ---- GATTGCGGAGTCGCGTG CGCCGCGTCAATCA
C.phaeocauiiS a CGCGCGTCAATCATTTGC [230]
C.phaeocauliS b GCCCTCCA ---- GAGACYCTGTGTGAT GATTGCGGCGC
GTCCTCCA ---- GAGACCCTGTGTGAT GATTGCGAGTCGCGTGCGCG TCAATCA TTTGC [2301
C.amarisSima a
GTCCTCAA ---- GAGACCCTGTGTGAT GATTG TCGCT CGCCGCGTCAATCATTTGC [228]
C. amarissima b [246]
C. auran tiaca [230]
C. heyrieana a
[234]
C. heyneana b
C. longal a GCCCTCAATAAAGAGACCCTGTGTGATTGTGATTGCGGAGCCGCGCGGCGCCGCGTCTCATTTGC [238]
[228]
C. .longal b
C. longa2 a GCCCTCAATCAAGAGACCCTGTGTGATTGATGATTGCGGAGCCGCGCGAGCGCCGCGTCTCATTTGC [2381
GYCCTCAA ---- GAGACYSTGTGTGAT GATTGCGG CGTCAATCATTTGC [2281
C. .longa2 b
[230]
C.amada a
I1
GTCCTCAA ---- GAGACCCTGTGTGAT GATTGC AGTC CGT NGCGTCATCATTT [228]
C. amada b
200 210 220 230 240 250
190
. 9. . . .0 1 .2
8
C.zedoarial a
C.zedoarial b GTCCTCAATCAGAGACCCTGTGTGAT ---- GATTGCNGAGGTGCGTGAIGCGGTCCGTCTCATTTGC [2321
[230)
C. zedoaria2a
C. zedoaria2b GTCCTCAATCAAGAGACCCTGTGTGAT ---- GATTGCGGAGTCGCGTGWGCGCCGCGTCTCATTTGC [232]
[2301
C. zedoaria3a
C. zedoaria3b GTCCTCCATCAAGAGACCCTGTGTGAT ---- GATTGCGGAGTCGCGTGAGCGCCGCGTCTCATTTGC [2321
C.zedoaria cf. a GTCCTCATCGAGACCCTGTGTGAT ---- GATTGCGGAGTCGCGTGAGCGCCGCGTCTCATTTGC [232]
[230)
C.zedoaria cf. b
C. zanthorrhizala GTCCTCCA ---- GAGACCCTGTGTGAT ---- GATTG0GGTCGCGTGCGG0GTCTCATTTGC [230]
[2321
C. zanthorrhizalb
C. zanthorrhiza2a
NJ
Lu
[2321
C. zanthorrhiza2b [230)
C.soloensLs a
C.soloens3.S b GYCCTCAATAAAGAGACCCTGTGTRATTGATGATTGCGGAGCCGCGCGAGCGCCGCGTCTCATTTGC [238]
[234]
C.aromatica a
C. aromatica b GCCCTCAAGCAAGAGACCCTGCGTGAT ---- GATTGCGGAGYCGCGCACGCCGCG__TCTCATTTGC [2361
Notes:
Numbers in bold italic indicate number and position of iridel polymorphisms. AJTIG/C in bold shows insertion polymorphisms within
one individu correspond with species name in bold font. Hypens indicate alignment gaps, while hypens in bold shows deletion
polymorphisms within one individu correspond with species name in bold font. Uncertain nucleotide states are coded based on PAUP
conventions (Swofford 1993) as follows: K=G/T, M=A/C, R=A/G, S=C/G, W=AJT, Y=CIT, N=AJT/GIC. a and b after species name
indicate sequence copies a and b which were obtained after inspecting and editing the electropherogram and were not the results of
cloning. Square brackets at the end of sequences show the real spacer length of ITS2 region.
0
C)
C/)
APPENDIX 7 Sequence data matrix (displayed from 5' to 3') of aligned ITS2 region of 32 accessions
representing 27 taxa of Zingiberaceae after polymorphic sequences combined.
40 50 60 70 80 90
10 20 30
Taxon ITS2 **
1 . .
. .23 45. .
Ca. gracilis
Ca. spi ca ta
R. a uricula ta AT --- CGTCGCTTTTGCTCCATGCATTGCTGGTGTCGAGCG ------ GAAATTGGCCTCGTGTGTCCTC--GGGCACAGTCGGTTGAAGA
R. schneideriana
St. jnvolucra tus
Sm. supraneanae
C.parviflora
C. thorelii
C. roscoeana
C. alisma tifolia
C. gracillima
t C. ecoma ta ATAGTCGGTCGAAGA
C. harmandii
C. cf. a
C.petioia ta
C. ochrorrhiza
C. aeruginosa
C.phaeocaulis
C. amarissima
C. aurantiaca ATTGCCGCCGCTTTTGCTCCATGCTTTATTAGCATTGAGCAGCGCGTTGGCCCCGTGTGCCCTCGGGCACAGTCGGTCGGA
C. heyneana
CGTCGCTTTTGCTCCATGCTTTGTCGGCATTGAGCG GGGCAC.GTCGGTCGAAGA
C.l ongal
AT___YGTCGCTTTTGCTCCATGCTYYGTYGG0ATTG0_0GTT0CCCGTGTT GGGCACP.GTCGGTCGAAGA
C. longa2
C. amada
C. zedoarial
C. zedoaria2
C. zedoaria3
C. zedoaria cf.
C. zanthorrhizal
C. zanthorrhiza2
C. so1oenss
C. aroma tica (1D
120 130 140 150 160 170 180
100 110
Taxori . . . . . .
. 7 ** .
. 8* ** **
6 . . . . .
Ca. gracilis
Ca. spica ta
R. auricula ta
GTGGG-TAGTCCGCAGTCGTCGGGCACGATGGGTGTTGGTCGCCGTGAGCGAGAACAGAACGT -----
----- CCCCGTCGC--TTTAGGATT
R. schneideriana
St. involucra tus
Sm. supraneanae
C.parviflora
C. thoreiii
C. roscoeana
C. alisma tifolia
C. gracillima
C. ecoma ta
C. harmandii
C. cf. a
Fj C.petioia ta
00 C. ochrorrhiza
C. aeruginosa
GYGGG_TAGTYGGTAATCGTCGAGCACGATGGACGTTT0GT0G0GOTGCGT CCTCGTCGT--TTTGGGATGW
C.phaeocaulis
GTGGG-TAGTCGRTAATCGTCGAGCACGAYGGACGTTGGTCGTCGCGAGCGAGAACTGAACGTCG CCTCGTCGT--TTGGATGA
C. amarissima
C. auran tiaca GTGGGGTAGCCGGTAGTCGTCGAGCACGATGGATGTTGGTCGTCACGAGCGAGCTGCATCGT__CCTTGTCGTCGTTTCGGCGA
C. heyneana
C. longal
C. ion ga2
CCTCGTCGT--TGGATGA
C. amada
GTGGG_TAGTCGGYAATCGTCGGCACGATGGACGTTGGT0GT0G0G0GcTGCGT CCTCGTCGT--TTGGGATGA
C. zedoarial
GTGGG_TAGTCGGTAATCGTCGAGCACGATGGA0GTTGGTCGT0G0G CTT CCTCGTCGTTTTGGGATGA
C. zedoaria2
GTGGG_TAGTCGGTAATCGTCGAGCACGATGGA0GTTG0T0GTcCTPT CCTCGTCGT--TTT-GGGATGA
C. zedoaria3
GTGGG-TAGTCGGTAATCGTCGAGCACGATGGACGTTGGTCGTCGCGAGCGAGAACTGAACGTCGT CCTCGTCGT--ITGGGATGA
C. zedoaria cf.
AGO CTGTOG CCTCGTCGT--TTT-GGGATGA
C. zanthorrhza1 GTGGG_TAGTCGGYAATCGTCGAGCACGATGGACGTTGGT0GT
GO CTGAACGTCG CCTCGTCGT-TTTGGGATGA
C. zanthorrhiza2 GYGGG_TAGTCGGYAATCGTCGAGCACGATGGACGTTGGT0GT000
GYGGG_TAGTCGGTAATCGTCGAGCACGATGGA0GTTGGT00T0G0G0CTGCGTCG CCTCGTCGTTTTGGGATGA
C. soloensis
GCGGG-TAGTCGGCAATCGTCGAGCACGATGGACGTTGGTCGTCGCGAGCGAGAACTKAACGTCGT -- CCTCGTYGT --TTT-GGGATGA
C. aroma tica
0
0)
190 200 210 220 230 240 250
Taxon . . 1 . . .
** * ****
9 Q *******
Ca. gracilis GTCCTCAA ---- GAGACCCTGTGTGAT ------- TGTGATGTCGTGTGAAGTGCCGTGTCCATCAMTTGT
Ca. spica ta GTCCTCAA ---- GAGACCCTGTGTGAT ------- TGTGATGTCGTGTGAAGTGCCGTGTCCATCATTGT
R. auricula ta TTCCTCAA ---- GAGACCCCGTGTGAT ------- TGTGATGCGGTGTGAAGCCCCCTGTCCATCATTGT
R. schneideriana GTCCTCAA ---- GAGACCCCGTGTGAT ------- TGTGATGTCGTGCGAAGTGCCCTGTCCATCA"ATTGT
St. involucra tus GACCTCAA ---- GAGACCCTGTGTGAT ------ TTGCGGAGTCGGGTGAAGTGCCGTGTCCATCATTGT
Sm. supraneanae GTCCTCAA ---- GAGACCCTGTGTGAT ------ TTGCGGAGTCGGACGAAGTGCCGTGTCAATCA"TTTGT
C.parviflora GTCCTCAA ---- GAGACCCTGTGTGAT ------- TGTGATGTCGTGTGAAGTGCCGTGTCCATCAJTTGT
C. thorelii GTCCTCAA ---- GAGACCCTATGTGAT ------- TGCAGAGTCGGACGAAAGCGCTGTGTCAATCATCATTTGC
C. roscoeana
C. alismatifolia GTCCTCAA ---- GAGACCCTACGTGAT ------- TGCAGAGTCGGATGAAAGCGCTGTG"TCAATCATCATTCGC
C. gracillima GTCCTCAA ---- GAGACCCTATGTGAT ------- TGCAGAGTCGGACGWGCGCTGTGTCAATCATCATTTGC
C. ecoma ta GTCCTCAA ---- GAGACCCTGTGTGAT ------- TGCGGAGTCGGTTGAAGTGCCGTGTCAATCATTTGT
C. harmandii GTCCTCAA ---- GAGACCCTATGTGAT ------- TGCAGAGTCGGATGAGCGCTGTGTCATCATCATTTGC
C. cf.
C.petioia ta
rn
190 200 210 220 230 240 250
Taxon 1 .
9 Q ******* ** * ****
C. ochrorrhiza
C. aeruginosa
C.phaeocaulis
C. amarissima
C.aurantiaca
C. heyneana GTCCTCCA ---- GAGACCCTGTGTGAT SATTGCGGAGTCGCGTGAAGCGCCGCG--TCAATCA--TTTGC
C.lon gal GYCCTC GAGACCCTGTGTGAT GATTGCGGAGYCGCGYGAAAGCGCCGYGTCAATCA- -- -TTTGC
C. ion ga2 GYCCTC GAGACYSTGTGTGAT GATTGCGGAGYCGCGYGAAGCGCCGCG -- TCATCA"TTTGC
C. amada GTCCTCMA ---- GAGACCCTGTGTGAT ---- GATTGCGGAGTCNCGTGAG0TCTCA ---- TTTGC
C. zedoarial GTCCTCM GAGACCCTGTGTGAT____GATTGCNGAGKYKCGTGAAARCGSYC0G-T0T07----TTTGC
IQ C.zedoaria2 GTCCTCM GAGACCCTGTGTGAT- -- -GATTGCGGAGTCGCGTGA AGCGCCGCG--TCAATCA-- - -TTTGC
0 C.zedoaria3 GTCCTCM GAGACCCTGTGTGAT ---- GATTGCGGAGTCGCGTGAGCGCCG0GTCT ---- TTT
C.zedoaria cf. GTCCTCMA.
C. zanthorrhizal GTCCTCCA ---- GAGACCCTGTGTGAT SATTGCGGAGTCGCGTGAAGCGCCGCGTCAATCATTTGC
C. zanthorrhz.za2 GTCCTCCA ---- GAGACCCTGTGTGAT SATTGCGGAGTCGCGTGAAAGCGCCGCG -TCAATCA- -- -TTTGC
C. soloens.is GYCCTCM GAGACCCTGTGTRIAT GRTTGCGGAGYCGCGYGAAGCGCCGCGTCAATCA"TTTG 0
C. aroinatica GCCCTC GAGACCCTGCGTGAT ---- GATTGCGGAGYCGCGCGAAGCGCCGCG:TCT ---- TTT
Numbers in bold italic indicate the number and position of alignment gaps. shows indel polymorphisms (correspond with species
name in bold font). Hypens indicate alignment gaps. Uncertain nucleotide states are coded based ort AUP conventions (Swofford
1993) as follows: K=G/T, M=A/C, R=A/G, S=C/G, W=A/T, Y=C/T, N=AITIG/C. * in bold indicates nucleotide sites which were
excluded from part of phylogenetic analysis.
rn
Cn
APPENDICES
261
APPENDICES
262
APPENDICES
#41 MA (BO). Wonogiri, Kethu Forest, Marlina Ardiyani #47MA (BO). Karang
Anyar, Dusun Talpitu, Desa Ngemplak, Kecamatan Karang Pandan, Marlina Ardiyani
#55MA (BO). Ngawi, Kampung Tambak Selo, Marlina Ardiyani #64MA (E). Ngawi,
Kampung Tambak Selo, Marlina Ardiyani 965MA (BO). Ngawi, Kampung Tambak
Selo, Marlina Ardiyani #66MA (E). Blora, Desa Getas, Marlina Ardiyani #70MA
(BO). Randublatung, Kampung Banaran, Marlina Ardiyani #71 MA (E). Tempoeran,
Beumée #4989 (BO). Purworedjo, Heyne #AcNo 166927 (BO). Wonogiri, Tukluk,
Harini #86 (L). Papua New Guinea: Musgrave, along river, Nagata #3915 (E).
Altitude range: 50- 610 m Specimen examined. Heyne 691, s.d., Java: Bogor (L);
l-leyne 683, s.d., Java: Bogor (L); no collector 50, s.d., Java: Solo, Temu glenyeh (L);
263
APPENDICES
41267 (K). Sarawak: Rumah Gerasi, Nanga Ju, Sungai Mujok, near Julau, Rantai
Jawa #S67309 (E). Sarawak: Kampung Silantek, 85th miles Simanggang Road, 2nd
Division, Ilias bin Pale #S.42639 (E). Sarawak: Rumah Ubong, Sungai Balang, Nanga
Gaat, Kapit, 7th Division, Lee, Awa #S.50038 (E). Sarawak: Rumah Ubong, Sungai
Balang, Nanga Gaat, Kapit, 7th Division, Lee, Awa #S.50039 (E). Sarawak: Kampong
Kuala Tellian, near Mukah, Kandau Jenang #S581 15 (E). Sarawak: Kampung
Gumbang, Runi, Lai Shak Teck #S67409 (E). Singapore: Bukit Timah, #11362 (K).
Sri Lanka: Sinharaja, Burtt 96803 (E). Bopathella Falls, Marlina Ardiyani #28 (E).
Galla, Dubuc s.n. (E).
Thailand: Hat Yai, Trang, Prince Songkla University, Newman #59 (E, E).
Tripagodas, Burmese border, Bloembergen #48 (K). Mae Mawh, LAMPANG, Mae
Mawh Lignite mine area, Maxwell #90-598 (E). Chiang Mai, summit of Doi Miang
Awo, Maxwell #92-518 (E). Hin Dat, Put 939 (K). Muang Ngao, Lampang, Put 93998
(K). Aw Ong Kang, Geesink, Hattink, Phengklai #6624 (K).
264
APPENDICES
Curcuma indeterminate
Exsiccatae:
Jawa: Randoeblatoeng, Ottens #688 (K, L). Djukongdjukong, Backer #27587 (BO).
Puiau Kangean, Backer #30009 (BO). Buitenzorg, Botanic Garden, Heyne
#AcNo008261 I (BO). Gombong, Heyne #AcNo0082617 (BO). Mangkang, Leeuwen
s.n. (L, BO); Randoeblatoeng, Koorders #4225713 (BO).
265
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